Figure 7B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w . Sections were stained with anti - Iba1 or anti - CD11b antibodies ( red ) . Scale bar , 100 µm . C Quantification of microglial number ( n = 5 animals per group , one - way ANOVA ) . Note that Irf8 - / - mice were analyzed by CD11b immunostaining , while other strains were analyzed by Iba1 staining . D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) and anti - cleaved caspase - 3 ( blue ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Arrows indicate cleaved caspase - 3 + apoptotic cells . Insets are higher magnification images around apoptotic cells . Scale bar , 20 µm and 5 µm ( insets ) . E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice . 3D images reconstructed from confocal images were analyzed ( n = 36 - 50 cells from five animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) . F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice ( n = 36 - 50 cells from five animals per group , one - way ANOVA with post hoc Tukey ' s test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w. Sections were stained with anti-Iba1 or anti-CD11b antibodies (red). Scale bar, 100 µm. C Quantification of microglial number (n = 5 animals per group, one-way ANOVA). Note that Irf8-/- mice were analyzed by CD11b immunostaining, while other strains were analyzed by Iba1 staining.  D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets). E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice. 3D images reconstructed from confocal images were analyzed (n = 36-50 cells from five animals per group, Kruskal-Wallis test with post hoc Dunn's test). F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice (n = 36-50 cells from five animals per group, one-way ANOVA with post hoc Tukey's test).


Figure 1 ( C ) pS65 - Ub ( left ) and total Ub ( right ) immunoblots of mitochondria - enriched fractions from wild - type flies treated 3 days with either paraquat or vehicle ( sucrose ) . ( D ) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days . T , total lysate ; N , nuclear - enriched fraction ; M , mitochondria - enriched fraction ; S , post - mitochondrial supernatant . ( E ) Immunoblots for the indicated antibodies of ( T ) total lysates or ( M ) mitochondria - enriched fractions from fly heads ( with proboscises removed ) and bodies ( thoraces and abdomens ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) pS65-Ub (left) and total Ub (right) immunoblots of mitochondria-enriched fractions from wild-type flies treated 3 days with either paraquat or vehicle (sucrose).(D) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days. T, total lysate; N, nuclear-enriched fraction; M, mitochondria-enriched fraction; S, post-mitochondrial supernatant.(E) Immunoblots for the indicated antibodies of (T) total lysates or (M) mitochondria-enriched fractions from fly heads (with proboscises removed) and bodies (thoraces and abdomens).


Figure 2 ( C ) pS65 - Ub immunoblot of mitochondria - enriched fractions following the paraquat pulse - chase assay in wild - type and park - / - flies . UT , untreated ; PQ , 1 - day paraquat treatment ; Recovery , flies removed from paraquat and returned to normal food . Asterisk ( * ) denotes non - specific band . ( D ) pS65 - Ub lane densitometry analysis of n = 3 independent replicates from ( C ) , expressed relative to the most intense lane signal in each blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) pS65-Ub immunoblot of mitochondria-enriched fractions following the paraquat pulse-chase assay in wild-type and park-/- flies. UT, untreated; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food. Asterisk (*) denotes non-specific band. (D) pS65-Ub lane densitometry analysis of n = 3 independent replicates from (C), expressed relative to the most intense lane signal in each blot.


Figure 3 ( A - B ) pS65 - Ub immunoblot of whole - animal lysates from untreated flies of the indicated genotypes . park RNAi was induced by da - GAL4 ( ubiquitous ) , Mef2 - GAL4 ( muscle ) or nSyb - GAL4 ( neurons ) . ( C ) pS65 - Ub immunoblot of lysates from bodies or heads ( without proboscises ) , as indicated , from park - / - animals . ( E ) Higher magnification image of pS65 - Ub positive structures in flight muscle counterstained for ATP5A . Scale bar = 5 μm . Chart shows quantification of ATP5A levels of pS65 - Ub - positive ( + ve ) versus - negative ( - ve ) mitochondrial structures from 3 biological replicates ( 4 - 7 images per animal ; average per animal shown in open circles ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-B) pS65-Ub immunoblot of whole-animal lysates from untreated flies of the indicated genotypes. park RNAi was induced by da-GAL4 (ubiquitous), Mef2-GAL4 (muscle) or nSyb-GAL4 (neurons). (C) pS65-Ub immunoblot of lysates from bodies or heads (without proboscises), as indicated, from park-/- animals.(E) Higher magnification image of pS65-Ub positive structures in flight muscle counterstained for ATP5A. Scale bar = 5 μm. Chart shows quantification of ATP5A levels of pS65-Ub-positive (+ve) versus -negative (-ve) mitochondrial structures from 3 biological replicates (4-7 images per animal; average per animal shown in open circles).


Figure 5 ( A ) pS65 - Ub immunoblot of whole - animal lysates from wild - type , Atg5 - and park - / - animals harvested at the indicated ages . ( B ) pS65 - Ub immunoblot of whole - animal lysates from young animals of the following genotypes : wild - type , Pink1 - , park - / - , park RNAi ( daG4 > UAS - park RNAi ) , Atg1 RNAi ( daG4 > UAS - Atg1 RNAi ) , Atg8a - .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) pS65-Ub immunoblot of whole-animal lysates from wild-type, Atg5- and park-/- animals harvested at the indicated ages.(B) pS65-Ub immunoblot of whole-animal lysates from young animals of the following genotypes: wild-type, Pink1-, park-/-, park RNAi (daG4>UAS-park RNAi), Atg1 RNAi (daG4>UAS-Atg1 RNAi), Atg8a-.


Figure 6 ( A - G ) Representative images of pS65 - Ub immunostaining false - coloured for intensity ( Fire LUT ) in muscle segments 6 / 7 from wandering L3 larvae of the following genotypes : ( A ) wild type , ( B ) Pink1 - , ( C ) park - / - , ( D ) Atg5 - , ( E ) Atg8a - , ( F ) Atg5 - ; park - / - , ( G ) Atg8a - ; park - / - . ( H ) Quantification of number of pS65 - Ub puncta from A - G displaying mean + / - SEM from the indicated number of animals ( n = 3 - 5 as indicated ) . ( I ) Puncta volume from C - G expressed as mean + / - SEM ( n = 4 - 5 animals ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-G) Representative images of pS65-Ub immunostaining false-coloured for intensity (Fire LUT) in muscle segments 6/7 from wandering L3 larvae of the following genotypes: (A) wild type, (B) Pink1-, (C) park-/-, (D) Atg5-, (E) Atg8a-, (F) Atg5-; park-/-, (G) Atg8a-; park-/-. (H) Quantification of number of pS65-Ub puncta from A-G displaying mean +/- SEM from the indicated number of animals (n = 3-5 as indicated). (I) Puncta volume from C-G expressed as mean +/- SEM (n = 4-5 animals).


Figure 7 ( A ) pS65 - Ub immunoblot of whole - animal lysates following paraquat pulse - chase assay of Atg5 - flies with parkin overexpression ( OE ) ( Atg55cc5 daG4 > UAS - park ) compared with Atg5 - flies overexpressing mitochondrially targeted GFP ( Atg55cc5 daG4 > UAS - mito - HA - GFP ) . UT , untreated flies ; PQ , 1 - day paraquat treatment ; Recovery , flies removed from paraquat and returned to normal food . ( B ) pS65 - Ub lane densitometry , normalised to total protein ( RevertTM stain ) , of n = 3 independent replicates of ( A ) , expressed as pS65 - Ub intensity relative to the most intense band in each blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) pS65-Ub immunoblot of whole-animal lysates following paraquat pulse-chase assay of Atg5- flies with parkin overexpression (OE) (Atg55cc5 daG4>UAS-park) compared with Atg5- flies overexpressing mitochondrially targeted GFP (Atg55cc5 daG4>UAS-mito-HA-GFP). UT, untreated flies; PQ, 1-day paraquat treatment; Recovery, flies removed from paraquat and returned to normal food.(B) pS65-Ub lane densitometry, normalised to total protein (RevertTM stain), of n = 3 independent replicates of (A), expressed as pS65-Ub intensity relative to the most intense band in each blot.


Figure 1 ( B ) Western blot revealed with anti - I - Abβ JV2 antibody . WCL : whole cell lysate of MutuDC + pRBC ; S0 : supernatant following incubation with protein - G beads ; S1 : 1st wash of protein - G beads ; IP MHC II : eluates from Y - 3P - immunoprecipitated MHC II , boiled or not . Representative of 2 independent experiments . ( C ) Venn diagram of unique peptides from P . berghei ( left ) and Mus musculus ( right ) identified in the experiments performed with PbK pRBC ( blue ) and PbA pRBC ( orange ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Western blot revealed with anti-I-Abβ JV2 antibody. WCL: whole cell lysate of MutuDC + pRBC ; S0: supernatant following incubation with protein-G beads ; S1: 1st wash of protein-G beads ; IP MHC II: eluates from Y-3P-immunoprecipitated MHC II, boiled or not. Representative of 2 independent experiments.(C) Venn diagram of unique peptides from P. berghei (left) and Mus musculus (right) identified in the experiments performed with PbK pRBC (blue) and PbA pRBC (orange).


Figure 2 ( B ) Gating strategy to calculate the proportion of CD11a + CD49d + activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC , PbA pRBC , OVA peptide ( IR17 ) or ETRAMP10 . 2 peptide ( NL17 ) . ( C ) Percentage of IFN / TNF - double producing cells among activated CD4 T cells for each peptide . Basal level with MutuDC alone was subtracted . C , D : Data show the mean + / - SEM . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 0098 ; TIM14 , P = 0 . 078 ; ENO , P = 0 . 016 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 0039 ; EF1α , P = 0 . 0039 ; M1 , P = 0 . 016 ; MSP1 , P = 0 . 031 ; LDH , P = 0 . 062 ; ETRAMP , P = 0 . 001 ; ATPSYN , P = 0 . 25 ; END70 , P = 0 . 25 ; PDI , P = 0 . 16 ; GAPDH . 2 , P = 0 . 99 ; pRBC , P = 0 . 001 . N = 6 mice for ATPSYN , END70 , PDI , GAPDH . 2 , N = 11 mice for all other peptides , pooled from 3 replicates . ( D ) Hierarchy of immunodominance , depicted as the percentage of each peptide - specific response with respect to the total parasite - specific response obtained with PbA pRBC - loaded MutuDC . C , D : Data show the mean + / - SEM . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 0098 ; TIM14 , P = 0 . 078 ; ENO , P = 0 . 016 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 0039 ; EF1α , P = 0 . 0039 ; M1 , P = 0 . 016 ; MSP1 , P = 0 . 031 ; LDH , P = 0 . 062 ; ETRAMP , P = 0 . 001 ; ATPSYN , P = 0 . 25 ; END70 , P = 0 . 25 ; PDI , P = 0 . 16 ; GAPDH . 2 , P = 0 . 99 ; pRBC , P = 0 . 001 . N = 6 mice for ATPSYN , END70 , PDI , GAPDH . 2 , N = 11 mice for all other peptides , pooled from 3 replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Gating strategy to calculate the proportion of CD11a+ CD49d+ activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC, OVA peptide (IR17) or ETRAMP10.2 peptide (NL17).(C) Percentage of IFN/TNF-double producing cells among activated CD4 T cells for each peptide. Basal level with MutuDC alone was subtracted. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.(D) Hierarchy of immunodominance, depicted as the percentage of each peptide-specific response with respect to the total parasite-specific response obtained with PbA pRBC-loaded MutuDC. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.


Figure 3 ( B ) IFN / TNF - double producing cells among activated CD4 T cells , after subtracting the basal level with MutuDC alone ( mean + / - SEM ) . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 027 ; TIM14 , P = 0 . 84 ; ENO , P = 0 . 84 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 71 ; EF1α , P = 0 . 001 ; M1 , P = 0 . 032 ; MSP1 , P = 0 . 23 ; LDH , P = 0 . 016 ; ETRAMP , P = 0 . 064 ; ATPSYN , P = 0 . 77 ; END70 , P = 0 . 097 ; PDI , P = 0 . 0052 ; GAPDH . 2 , P = 0 . 19 ; pRBC , P = 0 . 001 . N = 15 mice pooled from 3 replicates . ( C ) Blood parasitemia ( mean + / - SEM ) monitored by flow cytometry after inoculation of 5x104 PbA . GFP spz in B6 mice , treated ( open squares ) or not ( black squares ) with CQ at day 6 and 7 pi , in order to avoid ECM without blunting infection . Day 6 , P = 0 . 29 ; Day 7 , P = 0 . 0013 by multiple unpaired t - tests . ( D ) IFN / TNF - double producing cells among activated CD4 T cells , at day 14 pi following CQ treatment ( mean + / - SEM ) . Basal level with MutuDC alone was subtracted . GAPDH . 1 , P = 0 . 99 ; EF1α , P = 0 . 062 ; ETRAMP , P = 0 . 031 ; pRBC , P = 0 . 031 by paired non - parametric Wilcoxon tests in comparison with OVA peptide .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) IFN/TNF-double producing cells among activated CD4 T cells, after subtracting the basal level with MutuDC alone (mean +/- SEM). Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.027 ; TIM14, P = 0.84 ; ENO, P = 0.84 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.71; EF1α, P = 0.001 ; M1, P = 0.032 ; MSP1, P = 0.23 ; LDH, P = 0.016 ; ETRAMP, P = 0.064 ; ATPSYN, P = 0.77 ; END70, P = 0.097 ; PDI, P = 0.0052 ; GAPDH.2, P = 0.19 ; pRBC, P = 0.001. N = 15 mice pooled from 3 replicates.(C) Blood parasitemia (mean +/- SEM) monitored by flow cytometry after inoculation of 5x104 PbA.GFP spz in B6 mice, treated (open squares) or not (black squares) with CQ at day 6 and 7 pi, in order to avoid ECM without blunting infection. Day 6, P = 0.29 ; Day 7, P = 0.0013 by multiple unpaired t-tests.(D) IFN/TNF-double producing cells among activated CD4 T cells, at day 14 pi following CQ treatment (mean +/- SEM). Basal level with MutuDC alone was subtracted. GAPDH.1, P = 0.99 ; EF1α, P = 0.062 ; ETRAMP, P = 0.031 ; pRBC, P = 0.031 by paired non-parametric Wilcoxon tests in comparison with OVA peptide.


Figure 4 Figure 4 . cDC1 are superior to other spleen antigen - presenting cells for MHC II presentation of PbA and non - PbA antigens ( A ) Gating strategy for FACS - sorting pDC ( Lin - CD11clo B220 + Ly6C + ) , cDC1 ( Lin - CD11c + CD8α + ) , cDC2 ( Lin - CD11c + CD8α - ) and a mix of macrophages moDC ( Lin - CD11c - CD11b + Ly6C + ) from spleens of mice infected for 6 days with PbA pRBC . ( B ) I - Ab presentation of ETRAMP10 . 2 peptide by the indicated APC , measured with BEZ hybridomas . PbA pRBC - loaded MutuDC were used as controls . Representative of 2 experiments with 4 infected mice pooled per replicate . ( C ) Fold - change surface expression of I - Ab in cDC2 over cDC1 in day 6 - infected mice ( mean + / - SEM of 5 experiments ) . Difference is not significant based on unpaired t - test with Welch ' s correction . ( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( D ) I - Ab - AS15 presentation after exogenous addition of AS15 peptide at the indicated concentrations , assessed with the BTg01Z hybridomas . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen . ( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( E ) I - Ab - AS15 presentation by DC incubated with heat - killed T . gondii at the indicated ratio , assessed with the BTg01Z hybridomas . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen . ( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( F ) I - Ab - IR17 presentation by DC incubated with OVA - expressing E . coli at the indicated ratio , assessed with the OTIIZ hybridomas . PbA pRBC - loaded MutuDC were used as controls . D , E , F : representative of 2 experiments with 4 infected mice pooled per replicate . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen . ( G ) In vivo pRBC uptake by cDC1 ( XCR1 + CD172a - ) and cDC2 ( XCR1 - CD172a + ) analyzed at 6 days post - infection with PbA . GFP . Gates were positioned based on the auto - fluorescence of the cDC subset in day 6 PbA - infected mice . As GFP + events are rare , 107 events were acquired for each mouse . Symbols show the mean percentage + / - SEM ( N = 4 mice per group ) of GFP + cells in each DC subset . * , P = 0 . 0005 by unpaired t - test without assuming consistent SD . ( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( H ) I - Ab - NL17 presentation by DC incubated with PbA pRBC at a ratio of 5 : 1 , assessed with the BEZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen . ( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( I ) I - Ab - IR17 presentation by DC incubated with OVA - expressing E . coli at a ratio of 20 : 1 , assessed with the OTIIZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen . ( J ) Fold - change surface expression of I - Ab in cDC2 over cDC1 in naïve mice ( mean + / - SEM of 4 experiments ) . Difference is not significant based on unpaired t - test with Welch ' s correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 Figure 4. cDC1 are superior to other spleen antigen-presenting cells for MHC II presentation of PbA and non-PbA antigens (A) Gating strategy for FACS-sorting pDC (Lin- CD11clo B220+ Ly6C+), cDC1 (Lin- CD11c+ CD8α+), cDC2 (Lin- CD11c+ CD8α-) and a mix of macrophages moDC (Lin- CD11c- CD11b+ Ly6C+) from spleens of mice infected for 6 days with PbA pRBC. (B) I-Ab presentation of ETRAMP10.2 peptide by the indicated APC, measured with BEZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. Representative of 2 experiments with 4 infected mice pooled per replicate.(C) Fold-change surface expression of I-Ab in cDC2 over cDC1 in day 6-infected mice (mean +/- SEM of 5 experiments). Difference is not significant based on unpaired t-test with Welch's correction.(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (D) I-Ab-AS15 presentation after exogenous addition of AS15 peptide at the indicated concentrations, assessed with the BTg01Z hybridomas.Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (E) I-Ab-AS15 presentation by DC incubated with heat-killed T. gondii at the indicated ratio, assessed with the BTg01Z hybridomas. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (F) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at the indicated ratio, assessed with the OTIIZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. D,E,F: representative of 2 experiments with 4 infected mice pooled per replicate. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.(G) In vivo pRBC uptake by cDC1 (XCR1+ CD172a-) and cDC2 (XCR1- CD172a+) analyzed at 6 days post-infection with PbA.GFP. Gates were positioned based on the auto-fluorescence of the cDC subset in day 6 PbA-infected mice. As GFP+ events are rare, 107 events were acquired for each mouse. Symbols show the mean percentage +/- SEM (N = 4 mice per group) of GFP+ cells in each DC subset. *, P = 0.0005 by unpaired t-test without assuming consistent SD.(H,I) I-Ab presentation by cDC isolated from naïve mice. (H) I-Ab-NL17 presentation by DC incubated with PbA pRBC at a ratio of 5:1, assessed with the BEZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.(H,I) I-Ab presentation by cDC isolated from naïve mice. (I) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at a ratio of 20:1, assessed with the OTIIZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.(J) Fold-change surface expression of I-Ab in cDC2 over cDC1 in naïve mice (mean +/- SEM of 4 experiments). Difference is not significant based on unpaired t-test with Welch's correction.


Figure 5 ( B ) Gating strategy to assess efficiency of cDC1 depletion . ( C ) Percentage ( mean + / - SEM ) of CD8α + out of Lin - B220 - CD11c + MHC IIhi cells . * , P = 0 . 0043 by Mann - Whitney test . ( D ) FACS plots showing the proportion of CD11a + CD49d + CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC , PbA pRBC or ETRAMP10 . 2 peptide . ( E ) Proportion of CD11a + CD49d + CD4 T cells that co - produce IFN and TNF ( mean + / - SEM ) in response to MutuDC loaded with the indicated antigen , isolated from Karma mice treated ( open circles ) or not ( black circles ) with DT . Asterisks show significant differences between no DT and + DT group by multiple unpaired t - tests without assuming consistent SD . RBC , P = 0 . 18 ; GAPDH . 1 , P = 0 . 019 ; EF1a , 0 . 039 ; ETRAMP , P = 0 . 00054 ; OVA , P = 0 . 25 ; pRBC , P = 0 . 015 . ( F ) Absolute numbers of IFN + TNF + CD11a + CD49d + CD4 + T cells responding to the indicated stimuli ( mean + / - SEM ) . GAPDH . 1 , P = 0 . 0047 ; EF1a , 0 . 00039 ; ETRAMP , P = 0 . 00022 ; pRBC , P = 0 . 00019 by multiple unpaired t - tests without assuming consistent SD . ( G ) Blood parasitemia ( mean + / - SEM ) at day 4 and 6 pi in Karma mice treated ( open circles ) or not ( black circles ) with DT . Day 4 , P = 0 . 022 ; Day 6 , P = 0 . 77 by multiple unpaired t - tests without assuming consistent SD . C , E , F , G : No DT group , N = 5 mice ; + DT group , N = 6 mice . Representative of 3 independent experiments . ( H ) Absolute numbers of CD4 + T cells producing IFN , TNF or IL - 10 , either individually or in combination , following restimulation with MutuDC + PbA pRBC ( mean + / - SEM ) . These cells are called cytokine + in the next panels . * , P = 0 . 0023 by unpaired t - test without assuming consistent SD . ( I ) Multifunctionality analysis ( IFN , TNF , IL - 10 ) of cytokine + T cells responding to MutuDC loaded with PbA pRBC . ( J ) Proportion of IL - 10 + IFN - TNF - among cytokine + T cells , in response to MutuDC loaded with the indicated antigen ( mean + / - SEM ) . GAPDH . 1 , P = 0 . 32 ; EF1a , 0 . 16 ; ETRAMP , P = 8 . 5x10 - 5 ; OVA , P = 0 . 11 ; pRBC , P = 0 . 0015 by multiple unpaired t - tests without assuming consistent SD . H , I , J : N = 3 mice per group .

O O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Gating strategy to assess efficiency of cDC1 depletion.(C) Percentage (mean +/- SEM) of CD8α+ out of Lin- B220- CD11c+ MHC IIhi cells. *, P = 0.0043 by Mann-Whitney test.(D) FACS plots showing the proportion of CD11a+ CD49d+ CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC or ETRAMP10.2 peptide.(E) Proportion of CD11a+ CD49d+ CD4 T cells that co-produce IFN and TNF (mean +/- SEM) in response to MutuDC loaded with the indicated antigen, isolated from Karma mice treated (open circles) or not (black circles) with DT. Asterisks show significant differences between no DT and + DT group by multiple unpaired t-tests without assuming consistent SD. RBC, P = 0.18 ; GAPDH.1, P = 0.019 ; EF1a, 0.039 ; ETRAMP, P = 0.00054 ; OVA, P = 0.25 ; pRBC, P = 0.015.(F) Absolute numbers of IFN+ TNF+ CD11a+ CD49d+CD4+ T cells responding to the indicated stimuli (mean +/- SEM). GAPDH.1, P = 0.0047 ; EF1a, 0.00039 ; ETRAMP, P = 0.00022 ; pRBC, P = 0.00019 by multiple unpaired t-tests without assuming consistent SD.(G) Blood parasitemia (mean +/- SEM) at day 4 and 6 pi in Karma mice treated (open circles) or not (black circles) with DT. Day 4, P = 0.022 ; Day 6, P = 0.77 by multiple unpaired t-tests without assuming consistent SD. C,E,F,G: No DT group, N = 5 mice ; + DT group, N = 6 mice. Representative of 3 independent experiments.(H) Absolute numbers of CD4+ T cells producing IFN, TNF or IL-10, either individually or in combination, following restimulation with MutuDC+PbA pRBC (mean +/- SEM). These cells are called cytokine+ in the next panels. *, P = 0.0023 by unpaired t-test without assuming consistent SD.(I) Multifunctionality analysis (IFN, TNF, IL-10) of cytokine+ T cells responding to MutuDC loaded with PbA pRBC.(J) Proportion of IL-10+ IFN- TNF- among cytokine+ T cells, in response to MutuDC loaded with the indicated antigen (mean +/- SEM). GAPDH.1, P = 0.32 ; EF1a, 0.16 ; ETRAMP, P = 8.5x10-5 ; OVA, P = 0.11 ; pRBC, P = 0.0015 by multiple unpaired t-tests without assuming consistent SD. H,I,J: N = 3 mice per group.


figf1 ( a , b ) Changes in HEK293T cell cytoplasmic ( a ) and mitochondrial ( b ) [ Ca2 + ] in response to ionomycin ( 2 . 5 μM ) were simultaneously measured by fluo - 4 and rhod - 2 imaging , respectively . Each bar represents one target gene silenced with pooled siRNA . ( c ) qrtPCR of MCU , MCUR1 and MICU1 mRNA from mouse tissues ( n = 3 ; mean ± s . e . m . ) . ( d ) qrtPCR of MCUR1 mRNA from HEK293T cell clones ( n = 3 ; mean ± s . e . m . ) . KD , knockdown . Neg shRNA , negative shRNA . ( e ) qrtPCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone cells ( n = 3 ; mean ± s . e . m . ) . The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2 , respectively . ( f ) MCUR1 protein expression levels ( top ) and densitometric analysis ( bottom left ) ( n = 3 ; ± s . e . m . ) . Bottom right , Flag - tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA - resistant MCUR1 cDNA plasmid . WB , western blot . ( g , h ) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic ( green ) and mitochondrial ( red ) [ Ca2 + ] before , during and after ionomycin exposure . Scale bar , 20 μm . ( i - p ) Cytoplasmic ( green ) and mitochondrial matrix ( red ) [ Ca2 + ] responses in HEK293T ( i - l ) cells challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( i ) Wild - type HEK293T cells . ( j ) Cells expressing negative shRNA . ( k ) Clone shHK5 ( n = 4 ) . ( l ) Quantification of peak rhod - 2 fluorescence . * * P0 . 01 ( mean ± s . e . m . ) . ( i - p ) Cytoplasmic ( green ) and mitochondrial matrix ( red ) [ Ca2 + ] responses in HeLa ( m - p ) cells challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( m ) HeLa cells expressing negative shRNA . ( n ) Clone shHe2 . ( o ) Clone shHe2 re - expressing MCUR1 ( n = 3 ) . ( p ) Quantification of peak rhod - 2 fluorescence . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ) . ( q ) [ Ca2 + ] c and [ Ca2 + ] m signals evoked by ATP ( 100 μM ) and thapsigargin ( Tg , 2 μM ) were monitored simultaneously using fura - 2 / AM and mitopericam ( ipcam490 ) , respectively in control ( left ) and MCUR1 - knockdown ( right ) HeLa cells . [ Ca2 + ] c is calibrated in nanomolar concentrations ( black ) , whereas mitopericam fluorescence is inversely normalized to baseline ( F0 / F ; red ) . ( r ) Summary mean [ Ca2 + ] c and [ Ca2 + ] m peaks during ATP stimulation ( negative shRNA n = 29 ; MCUR1 knockdown n = 36 cells ) . * P0 . 05 ( mean ± s . e . m . ) . ( s ) Increase in bath [ Ca2 + ] ( Rfura2 ) and [ Ca2 + ] m ( Ripcam ) signals in response to CaCl2 ( 1 μM ) and InsP3 ( IP3 , 7 . 5 μM ) addition in permeabilized cells . Uncropped images of blots / gels are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a,b) Changes in HEK293T cell cytoplasmic (a) and mitochondrial (b) [Ca2+] in response to ionomycin (2.5 μM) were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. Each bar represents one target gene silenced with pooled siRNA.(c) qrtPCR of MCU, MCUR1 and MICU1 mRNA from mouse tissues (n = 3; mean ± s.e.m.).(d) qrtPCR of MCUR1 mRNA from HEK293T cell clones (n = 3; mean ± s.e.m.). KD, knockdown. Neg shRNA, negative shRNA.(e) qrtPCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone cells (n = 3; mean ± s.e.m.). The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2, respectively.(f) MCUR1 protein expression levels (top) and densitometric analysis (bottom left) (n = 3; ± s.e.m.). Bottom right, Flag-tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA-resistant MCUR1 cDNA plasmid. WB, western blot.(g,h) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic (green) and mitochondrial (red) [Ca2+] before, during and after ionomycin exposure. Scale bar, 20 μm.(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HEK293T (i-l) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (i) Wild-type HEK293T cells. (j) Cells expressing negative shRNA. (k) Clone shHK5 (n = 4). (l) Quantification of peak rhod-2 fluorescence. **P0.01 (mean±s.e.m.).(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HeLa (m-p) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n = 3). (p) Quantification of peak rhod-2 fluorescence. *P0.05, **P0.01 (mean±s.e.m.).(q) [Ca2+]c and [Ca2+]m signals evoked by ATP (100 μM) and thapsigargin (Tg, 2 μM) were monitored simultaneously using fura-2/AM and mitopericam (ipcam490), respectively in control (left) and MCUR1-knockdown (right) HeLa cells. [Ca2+]c is calibrated in nanomolar concentrations (black), whereas mitopericam fluorescence is inversely normalized to baseline (F0/F; red).(r) Summary mean [Ca2+]c and [Ca2+]m peaks during ATP stimulation (negative shRNA n = 29; MCUR1 knockdown n = 36 cells). *P0.05 (mean±s.e.m.).(s) Increase in bath [Ca2+](Rfura2) and [Ca2+]m (Ripcam) signals in response to CaCl2 (1 μM) and InsP3 (IP3, 7.5 μM) addition in permeabilized cells. Uncropped images of blots/gels are shown in Supplementary Fig. S6.


figf2Digitonin - permeabilized HeLa cells bathed in intracellular - like solution containing thapsigargin ( Tg ) were loaded with the ΔΨm indicator JC - 1 and the Ca2 + indicator Fura2FF , to which pulses of 10 μM Ca2 + were added before the addition of the mitochondrial uncoupler CCCP ( carbonyl cyanide m - chloro phenyl hydrazone ) . ( a - e ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] and ΔΨm in cells expressing negative shRNA ( Neg shRNA ; a ) , clone shHe1 ( b ) , clone shHe2 ( c ) , clone shHe2 re - expressing MCUR1 ( d ) and HeLa cells stably overexpressing MCUR1 ( e ) . Under similar conditions , 1 μM Ru360 was added before 10 μM Ca2 + pulses until the addition of CCCP . ( f - i ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] and ΔΨm in negative shRNA cells ( f ) , MCUR1 - knockdown clone shHe1 ( g ) and shHe2 ( h ) , and in control cells overexpressing MCUR1 ( i ) . ( j , k ) Negative shRNA ( j ) and MCUR1 - overexpressing ( k ) HEK293T cells were permeabilized with digitonin in intracellular - like medium containing thapsigargin and bath [ Ca2 + ] indicator Fura2FF , and then pulsed with 10 μM Ca2 + . After mitochondrial clearance of bath Ca2 + , Ru360 caused an elevation of bath [ Ca2 + ] , indicating that steady - state bath [ Ca2 + ] after the pulse was maintained by a balance of MCU - mediated Ca2 + uptake and CGP37157 ( 10 μM ) - sensitive Na + - Ca2 + exchanger - mediated extrusion . CCCP was added as indicated . The solid line is the mean ; shaded areas are ± s . e . m . ( n = 3 ) . ( l ) [ Ca2 + ] m efflux rate derived from j and k during the initial 60 s following Ru360 addition ( NS ; not significant ; n = 3 ) . Digitonin - permeabilized HeLa cells bathed in intracellular - like solution containing thapsigargin ( Tg ) were loaded with the ΔΨm indicator JC - 1 and the Ca2 + indicator Fura2FF , to which pulses of 10 μM Ca2 + were added before the addition of the mitochondrial uncoupler CCCP ( carbonyl cyanide m - chloro phenyl hydrazone ) . ( a - e ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] and ΔΨm in cells expressing negative shRNA ( Neg shRNA ; a ) , clone shHe1 ( b ) , clone shHe2 ( c ) , clone shHe2 re - expressing MCUR1 ( d ) and HeLa cells stably overexpressing MCUR1 ( e ) . Under similar conditions , 1 μM Ru360 was added before 10 μM Ca2 + pulses until the addition of CCCP .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca2+ indicator Fura2FF, to which pulses of 10 μM Ca2+ were added before the addition of the mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). (a-e) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in cells expressing negative shRNA (Neg shRNA; a), clone shHe1 (b), clone shHe2 (c), clone shHe2 re-expressing MCUR1 (d) and HeLa cells stably overexpressing MCUR1 (e). Under similar conditions, 1 μM Ru360 was added before 10 μM Ca2+ pulses until the addition of CCCP.(f-i) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in negative shRNA cells (f), MCUR1-knockdown clone shHe1 (g) and shHe2 (h), and in control cells overexpressing MCUR1 (i).(j,k) Negative shRNA (j) and MCUR1-overexpressing (k) HEK293T cells were permeabilized with digitonin in intracellular-like medium containing thapsigargin and bath [Ca2+] indicator Fura2FF, and then pulsed with 10 μM Ca2+. After mitochondrial clearance of bath Ca2+, Ru360 caused an elevation of bath [Ca2+], indicating that steady-state bath [Ca2+] after the pulse was maintained by a balance of MCU-mediated Ca2+ uptake and CGP37157 (10 μM)-sensitive Na+-Ca2+ exchanger-mediated extrusion. CCCP was added as indicated. The solid line is the mean; shaded areas are ±s.e.m. (n = 3).(l) [Ca2+]m efflux rate derived from j and k during the initial 60 s following Ru360 addition (NS; not significant; n = 3).Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca2+ indicator Fura2FF, to which pulses of 10 μM Ca2+ were added before the addition of the mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). (a-e) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in cells expressing negative shRNA (Neg shRNA; a), clone shHe1 (b), clone shHe2 (c), clone shHe2 re-expressing MCUR1 (d) and HeLa cells stably overexpressing MCUR1 (e). Under similar conditions, 1 μM Ru360 was added before 10 μM Ca2+ pulses until the addition of CCCP.


figf3 ( a , b ) Confocal images of HeLa cells transiently co - transfected for 48 h with GFP - tagged MCUR1 and DsRed - Mito plasmids ( a ) or mRFP - tagged MCUR1 and EYFP - Mito ( b ) . Scale bar , 20 μm . ( c ) Immunoblot analysis of Flag - tagged MCUR1 in the HeLa cell crude mitochondrial fraction , outer mitochondrial membrane ( OMM ) and mitoplasts , using antibodies against VDAC1 ( outer mitochondrial membrane protein ) , COII ( integral membrane marker ) and Flag . ( d ) Immunoblot analyses of mitochondria - containing pellet and cytosolic fractions from plasma - membrane - permeabilized HeLa cells . Permeabilized cells were treated with or without tBid ( 50 nM ) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified . Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min . These samples were probed using antibodies against HSP60 , OXA1 , Flag and MCUR1 . ( e ) Reciprocal co - immunoprecipitation ( IP ) of MCU - GFP and MCUR1 - Flag transiently expressed in COS7 cells . Representative of four independent experiments . WB , western blot . ( f ) Co - immunoprecipitation of MICU1 - Flag with MCU - GFP but not with MCUR1 - GFP transiently expressed in COS7 cells . Representative of four independent experiments . ( g ) Immunoprecipitation of LETM1 - GFP with anti - LETM1 failed to pull down MCUR1 - Flag in transiently transfected COS7 cells . ( h ) Immunoprecipitation with Flag antibody pulled down LETM1 - Flag or MCU - Flag transiently expressed in COS7 cells ( immunoprecipitation lanes 2 and 3 , lower panel ) , but only co - immunoprecipitated MCUR1 - V5 in the MCU - Flag - expressing cells ( immunoprecipitation lanes 4 versus 5 , upper panel ) , despite lower expression of MCU - Flag in MCUR1 - co - transfected cells ( compare MCU - Flag and LETM1 - Flag expression in MCUR1 - co - transfected cells in lysate lanes 4 and 5 , bottom panel ) . Representative of three independent experiments . Uncropped images of blots / gels are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a,b) Confocal images of HeLa cells transiently co-transfected for 48 h with GFP-tagged MCUR1 and DsRed-Mito plasmids (a) or mRFP-tagged MCUR1 and EYFP-Mito (b). Scale bar, 20 μm.(c) Immunoblot analysis of Flag-tagged MCUR1 in the HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (outer mitochondrial membrane protein), COII (integral membrane marker) and Flag.(d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma-membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified. Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1.(e) Reciprocal co-immunoprecipitation (IP) of MCU-GFP and MCUR1-Flag transiently expressed in COS7 cells. Representative of four independent experiments. WB, western blot.(f) Co-immunoprecipitation of MICU1-Flag with MCU-GFP but not with MCUR1-GFP transiently expressed in COS7 cells. Representative of four independent experiments.(g) Immunoprecipitation of LETM1-GFP with anti-LETM1 failed to pull down MCUR1-Flag in transiently transfected COS7 cells.(h) Immunoprecipitation with Flag antibody pulled down LETM1-Flag or MCU-Flag transiently expressed in COS7 cells (immunoprecipitation lanes 2 and 3, lower panel), but only co-immunoprecipitated MCUR1-V5 in the MCU-Flag-expressing cells (immunoprecipitation lanes 4 versus 5, upper panel), despite lower expression of MCU-Flag in MCUR1-co-transfected cells (compare MCU-Flag and LETM1-Flag expression in MCUR1-co-transfected cells in lysate lanes 4 and 5, bottom panel). Representative of three independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. S6.


figf4 ( a ) qrtPCR of MCU mRNA from wild - type and stable MCUR1 - overexpressing HeLa cells that were transiently transfected with scrambled siRNA or siRNA against MCU . * * * P0 . 001 ( mean ± s . e . m . ) . ( b ) [ Ca2 + ] m responses to histamine ( 100 μM ) in HeLa cells stably overexpressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA , and in stable MCUR1 - overexpressing HeLa cells transfected with MCU siRNA . After 48 h of siRNA transfection , cells were loaded with rhod - 2 and [ Ca2 + ] m responses were visualized by confocal microscopy . Solid lines are mean ; shaded regions are ± s . e . m . ; n = 3 . ( c ) Quantification of peak rhod - 2 fluorescence following histamine stimulation . * P0 . 05 , * * * P0 . 001 ( mean ± s . e . m . ; NS , not significant ; n = 3 ) . ( d ) qrtPCR of MCU mRNA from wild - type and MCUR1 - knockdown HeLa cells that were transiently transfected with MCU cDNA . * * P0 . 01 , * * * P0 . 001 ( mean ± s . e . m . ; n = 3 ) . ( e ) [ Ca2 + ] m responses to histamine ( 100 μM ) in wild - type and MCUR1 ( shHe2 ) - knockdown HeLa cells overexpressing MCU . Negative shRNA ( Neg shRNA ) and MCUR1 - shHe2 cells were used as controls . Solid lines are mean ; shaded regions are ± s . e . m . ; n = 3 . ( f ) Quantification of peak rhod - 2 fluorescence following histamine stimulation . * * * P0 . 001 ( mean ± s . e . m . ; n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) qrtPCR of MCU mRNA from wild-type and stable MCUR1-overexpressing HeLa cells that were transiently transfected with scrambled siRNA or siRNA against MCU. ***P0.001 (mean±s.e.m.).(b) [Ca2+]m responses to histamine (100 μM) in HeLa cells stably overexpressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA, and in stable MCUR1-overexpressing HeLa cells transfected with MCU siRNA. After 48 h of siRNA transfection, cells were loaded with rhod-2 and [Ca2+]m responses were visualized by confocal microscopy. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (c) Quantification of peak rhod-2 fluorescence following histamine stimulation. *P0.05, ***P0.001 (mean±s.e.m.; NS, not significant; n = 3).(d) qrtPCR of MCU mRNA from wild-type and MCUR1-knockdown HeLa cells that were transiently transfected with MCU cDNA. **P0.01, ***P0.001 (mean±s.e.m.; n = 3).(e) [Ca2+]m responses to histamine (100 μM) in wild-type and MCUR1 (shHe2)-knockdown HeLa cells overexpressing MCU. Negative shRNA (Neg shRNA) and MCUR1-shHe2 cells were used as controls. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (f) Quantification of peak rhod-2 fluorescence following histamine stimulation. ***P0.001 (mean±s.e.m.; n = 3).


figf5 ( a ) AMP / ATP ratios in stable HeLa cell lines stably expressing negative shRNA ( Neg shRNA ) , MCUR1 shRNA ( clone shHe2 ) or shHe2 with MCUR1 re - expressed . * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) . ( b ) O2 consumption rates ( OCR ) in stable HeLa cells expressing irrelevant shRNA , clone shHe2 , and clone shHe2 re - expressing MCUR1 , exposed sequentially to oligomycin ( i ) , FCCP ( ii ) and rotenone plus myzothiazol ( iii ) . ( c ) Basal and maximal O2 consumption rates in cells as described in b . * P0 . 05 ( mean ± s . e . m . ; n = 3 ) . ( d ) Western blot of phosphorylated and total AMPK ( top ) and densitometric analysis ( bottom ) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA ( clone shHe2 ) and clone shHe2 re - expressing MCUR1 . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) . ( e ) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA ( clone shHe2 ) and clone shHe2 re - expressing MCUR1 ( top ) and quantification of LC3 - II / ( LC3 - I + LC3 - II ) ( bottom ) expressed as fold increase over levels in cells expressing irrelevant shRNA . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) . ( f , g ) , The same as in d and e . Activation of AMPK ( f ) and autophagy ( * * P0 . 001 ; mean ± s . e . m . ; n = 3 ) ( g ) in the absence and presence of InsP3R inhibitor xestospongin B ( XeB ) . * P0 . 05 , * * P0 . 001 , NS , not significant ( mean ± s . e . m . ; n = 3 ) . Uncropped images of blots / gels are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) AMP/ATP ratios in stable HeLa cell lines stably expressing negative shRNA (Neg shRNA), MCUR1 shRNA (clone shHe2) or shHe2 with MCUR1 re-expressed. **P0.01 (mean±s.e.m.; n = 3).(b) O2 consumption rates (OCR) in stable HeLa cells expressing irrelevant shRNA, clone shHe2, and clone shHe2 re-expressing MCUR1, exposed sequentially to oligomycin (i), FCCP (ii) and rotenone plus myzothiazol (iii).(c) Basal and maximal O2 consumption rates in cells as described in b. *P0.05 (mean±s.e.m.; n = 3).(d) Western blot of phosphorylated and total AMPK (top) and densitometric analysis (bottom) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1. *P0.05, **P0.01 (mean±s.e.m.; n = 3). (e) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1 (top) and quantification of LC3-II/(LC3-I + LC3-II) (bottom) expressed as fold increase over levels in cells expressing irrelevant shRNA. *P0.05, **P0.01 (mean±s.e.m.; n = 3).(f,g), The same as in d and e. Activation of AMPK (f) and autophagy (**P0.001; mean±s.e.m.; n = 3) (g) in the absence and presence of InsP3R inhibitor xestospongin B (XeB). *P0.05, **P0.001, NS, not significant (mean±s.e.m.; n = 3). Uncropped images of blots/gels are shown in Supplementary Fig. S6.


Figure 2 ( A ) B cell development in Igh ∆ 890 / ∆ 890 mice . The relative frequencies of the indicated cell types were determined by flow cytometric analysis of bone marrow cells from Igh ∆ 890 / ∆ 890 and Igh + / + mice at the age of 3 - 4 weeks , indicating a 1 . 3 - fold increase of pro - B cells and a moderate decrease at subsequent B cell developmental stages in Igh ∆ 890 / ∆ 890 mice . Statistical data are shown as mean value with SEM . Data information : Statistical data were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Sidak correction ; A , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot ( A , corresponds to one mouse . ( B ) Two - color 3D DNA - FISH analysis of ex vivo sorted pro - B cells of the indicated genotypes with the RP23 - 340K14 ( red ) and RP24 - 275L15 ( green ) BAC probes ( Fig 1A ) . Representative images are show above . Dot plots ( below ) show the distances measured between the two DNA signals in individual Igh alleles ( 2 , 123 for Rag2 - / - , 1 , 514 for Igh ∆ 890 / ∆ 890 Rag2 - / - and 502 for Vav - Cre Pax5fl / fl ( Pax5 ∆ / ∆ ) Rag2 - / - pro - B cells ) together with the mean distance ( red bar ) and SEM determined for each genotype . In total , four independent DNA - FISH experiments were performed . Data information : Statistical data were analyzed by multiple t - tests ( unpaired one - way ANOVA ( Tukey post - hoc test ; B ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( C , VH gene recombination analysis in ex vivo sorted Igh ∆ 890 / ∆ 890 and Igh + / + pro - B cells , as determined by VDJ - seq experiments ( C ) The percentages of uniquely identified DJH and VDJH sequences are shown as mean percentage with SEM . Each dot corresponds to an independent VDJ - seq experiments performed with sorted pro - B cells from one mouse . Data information : Statistical data were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Sidak correction ; ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot C , corresponds to one mouse .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) B cell development in Igh∆890/∆890 mice. The relative frequencies of the indicated cell types were determined by flow cytometric analysis of bone marrow cells from Igh∆890/∆890 and Igh+/+ mice at the age of 3-4 weeks, indicating a 1.3-fold increase of pro-B cells and a moderate decrease at subsequent B cell developmental stages in Igh∆890/∆890 mice. Statistical data are shown as mean value with SEM. Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; A, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (A, corresponds to one mouse.(B) Two-color 3D DNA-FISH analysis of ex vivo sorted pro-B cells of the indicated genotypes with the RP23-340K14 (red) and RP24-275L15 (green) BAC probes (Fig 1A). Representative images are show above. Dot plots (below) show the distances measured between the two DNA signals in individual Igh alleles (2,123 for Rag2-/-, 1,514 for Igh∆890/∆890 Rag2-/- and 502 for Vav-Cre Pax5fl/fl (Pax5∆/∆) Rag2-/-pro-B cells) together with the mean distance (red bar) and SEM determined for each genotype. In total, four independent DNA-FISH experiments were performed. Data information: Statistical data were analyzed by multiple t-tests (unpaired one-way ANOVA (Tukey post-hoc test; B); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.(C, VH gene recombination analysis in ex vivo sorted Igh∆890/∆890 and Igh+/+ pro-B cells, as determined by VDJ-seq experiments (C) The percentages of uniquely identified DJH and VDJH sequences are shown as mean percentage with SEM. Each dot corresponds to an independent VDJ-seq experiments performed with sorted pro-B cells from one mouse. Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot C, corresponds to one mouse.


Figure 3 ( B ) Flow cytometric determination of the ratio of immature IgMb ( B6 ) to IgMa ( 129 ) B cells in the bone marrow of Igh ∆ 890 ( B6 ) / + ( 129 ) , IghP4V ( B6 ) / + ( 129 ) and control Igh + ( B6 ) / + ( 129 ) mice , which were generated by crossing Igh ∆ 890 / + , IghP4V / + and Igh + / + mice on the C57BL / 6 ( B6 ) background with Igh + / + mice of the 129 / Sv ( 129 ) strain . The rearranged Igh alleles of the C57BL / 6 and 129 / Sv strains give rise to the expression of IgMb and IgMa , respectively . The IgMb ( B6 ) to IgMa ( 129 ) ratio is shown as mean value with SEM ( right ) . Data information : Statistical data were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Sidak correction ; B , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot ( B , corresponds to one mouse . ( C ) Summary of the ratios of immature IgMb ( B6 ) to IgMa ( 129 ) B cells , determined for the indicated six mouse strains analyzed and shown as mean values with SEM . The ratio determined for control Igh + ( B6 ) / + ( 129 ) B cells was set to 1 . Data information : Statistical data were analyzed by multiple t - tests ( unpaired one - way ANOVA ( Tukey post - hoc test ; C , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot C , corresponds to one mouse . ( D ) Comparison of the VDJ - seq data obtained with pro - B cells of the Igh + / + ( black ) , Igh ∆ 890 / ∆ 890 ( white bars ) and IghP4V / P4V ( gray ) genotypes . The different VH genes ( horizontal axis ) are aligned according to their position in the Igh locus The usage of each VH gene ( vertical axis ) is shown as percentage of all VDJH and DJH recombination events determined for each pro - B cell type . The relative frequency of each VH gene is shown as mean value with SEM and is based on three independent VDJ - seq experiments for pro - B cells of each genotype .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Flow cytometric determination of the ratio of immature IgMb (B6) to IgMa (129) B cells in the bone marrow of Igh∆890(B6)/+(129), IghP4V (B6)/+(129) and control Igh+(B6)/+(129) mice, which were generated by crossing Igh∆890/+, IghP4V/+ and Igh+/+ mice on the C57BL/6 (B6) background with Igh+/+ mice of the 129/Sv (129) strain. The rearranged Igh alleles of the C57BL/6 and 129/Sv strains give rise to the expression of IgMb and IgMa, respectively. The IgMb (B6) to IgMa (129) ratio is shown as mean value with SEM (right). Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; B, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (B, corresponds to one mouse.(C) Summary of the ratios of immature IgMb (B6) to IgMa (129) B cells, determined for the indicated six mouse strains analyzed and shown as mean values with SEM. The ratio determined for control Igh+(B6)/+(129) B cells was set to 1. Data information: Statistical data were analyzed by multiple t-tests (unpaired one-way ANOVA (Tukey post-hoc test; C, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot C, corresponds to one mouse.(D) Comparison of the VDJ-seq data obtained with pro-B cells of the Igh+/+ (black), Igh∆890/∆890 (white bars) and IghP4V/P4V (gray) genotypes. The different VH genes (horizontal axis) are aligned according to their position in the Igh locus The usage of each VH gene (vertical axis) is shown as percentage of all VDJH and DJH recombination events determined for each pro-B cell type. The relative frequency of each VH gene is shown as mean value with SEM and is based on three independent VDJ-seq experiments for pro-B cells of each genotype.


Figure 4 ( A ) Presence of open chromatin and the active histone marks H3K27ac and H3K4me2 at the V8 . 8E enhancer in pro - B cells . The location of the VH8 - 8 gene , V8 . 8E enhancer ( dashed line ) and PAIR5 element are shown together with the mm9 genomic coordinates of mouse chromosome 12 . The VH8 - 8 gene with 500 bp of its upstream and downstream sequences ( indicated by violet overlay ) was inserted with the Floxin method at the deletion point of the Igh ∆ 890 allele to generate the IghV8 - 8

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Figure 4(A) Presence of open chromatin and the active histone marks H3K27ac and H3K4me2 at the V8.8E enhancer in pro-B cells. The location of the VH8-8 gene, V8.8E enhancer (dashed line) and PAIR5 element are shown together with the mm9 genomic coordinates of mouse chromosome 12. The VH8-8 gene with 500 bp of its upstream and downstream sequences (indicated by violet overlay) was inserted with the Floxin method at the deletion point of the Igh∆890 allele to generate the IghV8-8 allele


Figure 5 ( B , Flow - cytometric analysis ( B ) of GFP expression are shown for pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghP4GV / P4GV ( dark green , n = 16 ) , IghP4GV / + ( light green , n = 12 ) and control Igh + / + ( black , n = 13 ) mice . C ) geometric mean fluorescence intensity measurements ( MFI , C ) of GFP expression are shown for pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghP4GV / P4GV ( dark green , n = 16 ) , IghP4GV / + ( light green , n = 12 ) and control Igh + / + ( black , n = 13 ) mice . Geometric mean fluorescence intensity measurements ( C ) are shown as mean values with SEM . G ) Absence of GFP expression in DN ( CD4 - CD8 - ) and DP ( CD4 + CD8 + ) T cells , geometric mean fluorescence intensity measurements ( G ) of GFP expression in thymocytes from the indicated mice . Geometric mean fluorescence intensity measurements ( G ) are shown as mean value with SEM based on 12 ( Igh + / + ) , 11 ( IghP4GV / + ) or 12 ( IghP4GV / P4GV ) independent replicates .

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Figure 5(B, Flow-cytometric analysis (B) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/P4GV (dark green, n=16), IghP4GV/+ (light green, n=12) and control Igh+/+ (black, n=13) mice.C) geometric mean fluorescence intensity measurements (MFI, C) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/P4GV (dark green, n=16), IghP4GV/+ (light green, n=12) and control Igh+/+ (black, n=13) mice. Geometric mean fluorescence intensity measurements (C) are shown as mean values with SEM.G) Absence of GFP expression in DN (CD4-CD8-) and DP (CD4+CD8+) T cells, geometric mean fluorescence intensity measurements (G) of GFP expression in thymocytes from the indicated mice. Geometric mean fluorescence intensity measurements (G) are shown as mean value with SEM based on 12 (Igh+/+), 11 (IghP4GV/+) or 12 (IghP4GV/P4GV) independent replicates.


Figure 6 ( A , B ) Flow - cytometric analysis ( A ) and geometric mean fluorescence intensity measurements ( MFI , B ) of GFP expression are shown for pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghP4GV / + ( green , n = 6 ) , IghP4 ∆ Pax5GV / + ( yellow , n = 5 ) , IghP4 ∆ CtcfGV / + ( brown , n = 4 ) and control Igh + / + ( black , n = 4 ) mice . Geometric mean fluorescence intensity measurements ( B ) are shown as mean value with SEM . Data information : Each dot corresponds to one mouse . ( C , D ) Activation of the PAIR4 - V8 . 7E module in thymocytes upon loss of CTCF binding at PAIR4 , as shown by flow - cytometric analysis ( C ) and geometric mean fluorescence intensity measurements ( MFI , D ) of GFP expression in DN , DP , CD4 + and CD8 + thymocytes of IghP4 ∆ CtcfGV / + ( light brown , n = 5 ) and IghP4 ∆ CtcfGV / P4 ∆ CtcfGV ( dark brown , n = 6 ) mice compared with control Igh + / + ( black , n = 7 ) mice . Geometric mean fluorescence intensity measurements ( D ) are shown as mean value with SEM . Data information : Each dot corresponds to one mouse .

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Figure 6(A, B) Flow-cytometric analysis (A) and geometric mean fluorescence intensity measurements (MFI, B) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/+ (green, n=6), IghP4∆Pax5GV/+ (yellow, n=5), IghP4∆CtcfGV/+ (brown, n=4) and control Igh+/+ (black, n=4) mice. Geometric mean fluorescence intensity measurements (B) are shown as mean value with SEM. Data information: Each dot corresponds to one mouse.(C, D) Activation of the PAIR4-V8.7E module in thymocytes upon loss of CTCF binding at PAIR4, as shown by flow-cytometric analysis (C) and geometric mean fluorescence intensity measurements (MFI, D) of GFP expression in DN, DP, CD4+ and CD8+ thymocytes of IghP4∆CtcfGV/+ (light brown, n=5) and IghP4∆CtcfGV/P4∆CtcfGV (dark brown, n=6) mice compared with control Igh+/+ (black, n=7) mice. Geometric mean fluorescence intensity measurements (D) are shown as mean value with SEM. Data information: Each dot corresponds to one mouse.


Figure 7 ( B , C ) Flow - cytometric analysis ( B ) and geometric mean fluorescence intensity measurements ( MFI , C ) of YFP expression are shown for MPPs ( LSK ) , LMPPs , ALPs and BLPs from the bone marrow of Rosa26LSL - YPF / + IghCμCre / + ( violet ) and control Rosa26LSL - YPF / + Igh + / + ( black ) mice . Geometric mean fluorescence intensity measurements ( B ) are shown as mean value with SEM and have been normalized by setting the mean value obtained with Igh + / + cells to 1 . Data information : Each dot corresponds to one mouse . Flow - cytometric analysis ( E ) of GFP expression are shown for MPPs ( LSK ) , LMPPs , ALPs , BLPs , pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghEμG / + ( light blue ) , IghEμG / EμG ( dark blue ) and control Igh + / + ( black ) mice .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B, C) Flow-cytometric analysis (B) and geometric mean fluorescence intensity measurements (MFI, C) of YFP expression are shown for MPPs (LSK), LMPPs, ALPs and BLPs from the bone marrow of Rosa26LSL-YPF/+ IghCμCre/+ (violet) and control Rosa26LSL-YPF/+ Igh+/+ (black) mice. Geometric mean fluorescence intensity measurements (B) are shown as mean value with SEM and have been normalized by setting the mean value obtained with Igh+/+ cells to 1. Data information: Each dot corresponds to one mouse.Flow-cytometric analysis (E) of GFP expression are shown for MPPs (LSK), LMPPs, ALPs, BLPs, pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghEμG/+ (light blue), IghEμG/EμG (dark blue) and control Igh+/+ (black) mice.


Figure 1B , Same as in A , with the exception that cells expressing flag - praja2rm or praja2 deletion mutants ( praja21 - 530 , praja21 - 630 and praja2Δ530 - 630 ) were included in the analysis .

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Figure 1B, Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis.


Figure 2B . MST signal ( normalized fluorescence ) of P1 ( red curve ) , P2 ( green curve ) and P3 ( cyan curve ) plotted against TBC1D31 , at increasing concentrations of peptides . The threading modelled structure of the overlapping binding segment of praja2 ( praja2550 - 570 ) is shown . F . MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides : wild - type ( red curve ) , R948A - R951A ( violet curve ) and R957 - R959D - H960A ( orange curve ) .

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Figure 2B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550-570) is shown.F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve).


Figure 3D . Total lysates from mouse brain were immunoprecipitated with anti - TBC1D31 antibodies or with control IgG . The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies .

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Figure 3D. Total lysates from mouse brain were immunoprecipitated with anti-TBC1D31 antibodies or with control IgG. The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies.


Figure 4E . Same as in C , with the exception that cells were co - transfected with flag - OFD1 vector and with control siRNA ( siCNT ) or siRNA targeting praja2 . F . Quantitative analysis of the experiments shown in E . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 . G

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Figure 4E. Same as in C, with the exception that cells were co-transfected with flag-OFD1 vector and with control siRNA (siCNT) or siRNA targeting praja2. F. Quantitative analysis of the experiments shown in E. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05. G


Figure 5C . HEK293 cells transfected with siRNAs were serum deprived , pre - treated with cycloheximide and stimulated with FSK at different time point . Lysates were immunoblotted for endogenous praja2 and OFD1 . Hsp90 was used as loading control . D . Quantitative analysis of the experiments shown in C . A mean value ± SD of three independent experiments is shown . Student ' s t test * p < 0 , 05 ; * * < 0 , 01 versus sipraja2 ( + FSK ) and basal values ( 0 ) . E H . Immunoblot analysis of flag - OFD1 or flag - E97G mutant in HEK293 cells stimulated with FSK as in C . I . Quantitative analysis of the experiments shown in H . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 versus E97G mutant ( + FSK ) and basal values ( 0 ) .

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Figure 5C. HEK293 cells transfected with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control. D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0). E H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with FSK as in C. I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0).


Figure 6A . siRNA transfected HEK293 cells were serum deprived and stained for acetylated - tubulin , TBC1D31 and DRAQ5 . B . Quantitative analysis of the experiments shown in A . A mean value ± SD of four independent experiments is shown . Student ' s t test * p < 0 , 05 . C C . HEK293 cells expressing flag - OFD1 or flag - S735A were serum deprived and subjected to staining analysis for acetylated α - tubulin , flag and DRAQ5 . Images were collected by super - resolution microscopy ( upper panels ) . Where indicated , flag - OFD1 or flag - S735A was co - transfected with cilia - APEX - GFP vector ( middle panels ) . NIH3T3 expressing flag - OFD1 or flag - S735A were stained with anti - ARL13B and anti - flag antibodies . D , E . Statistical analysis of the experiments shown in D . A mean value ± SD of three independent experiments is shown . For each experimental group , a minimum of 40 cilia were analyzed . Student ' s t test p * * * < 0 , 001 . F F . HEK293 cells transiently transfected with flag - OFD1 or flag - S735A were serum deprived , treated with FSK ( 6 hours ) and stained for flag , acetylated - tubulin and DRAQ5 . G . Statistical analysis of the experiments shown in F . A mean value ± SD of three independent experiments is shown . Cells analysed for each experiment : 300 for NT , 80 for flag - OFD1 and 80 for flag - S735A . Student ' s t test , p * < 0 , 05 , p * * < 0 , 01 . H

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Figure 6A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5. B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05. C C. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were stained with anti-ARL13B and anti-flag antibodies. D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001. F F. HEK293 cells transiently transfected with flag-OFD1 or flag-S735A were serum deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5. G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01. H


Figure 7B . Confocal images of cilia of the neural tube cells in the wild - type , Ol - TBC1D31 KD , Ol - TBC1D31 KD + hTBC1D31 , wild - type hOFD1 , Ol - TBC1D31 KD + wild - type hOFD1 , hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735D and Ol - TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti - acetylated α - tubulin antibody ( green ) and DAPI ( blue ) .

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Figure 7B. Confocal images of cilia of the neural tube cells in the wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti-acetylated α-tubulin antibody (green) and DAPI (blue).


Figure 1A . Characteristics of IGR39 / IGR37 and WM115 / WM266 . 4 melanoma cell lines ( Upper Panel ) . Pigmentation of melanoma cells pellets ( lower panel ) . B . Growth curve of primitive IGR39 and metastatic IGR37 cell line ( N = 3 biological replicates ) . Data are mean ± SD . C . Phase - contrast images of IGR39 and IGR37 cells . Scale bar is 100µm . D . Analysis of IGR39 and IGR37 speed of migration by time - lapse microscopy .

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Figure 1A. Characteristics of IGR39 /IGR37 and WM115/WM266.4 melanoma cell lines (Upper Panel). Pigmentation of melanoma cells pellets (lower panel).B. Growth curve of primitive IGR39 and metastatic IGR37 cell line (N=3 biological replicates). Data are mean ±SD.C. Phase-contrast images of IGR39 and IGR37 cells. Scale bar is 100µm.D. Analysis of IGR39 and IGR37 speed of migration by time-lapse microscopy.


Figure 2B . Scatter plot of identified and quantified proteins in the secretome of primitive IGR39 and metastatic IGR37 . Red dots represent proteins that were significant with FDR < 0 . 05 , blue dots represent proteins with p - value < 0 . 05 . C . Scatter plot of identified and quantified proteins fin the secretome of primitive WM115 and metastatic WM266 . 4 . Red dots represent proteins that were significant with FDR < 0 . 05 , blue dots represent proteins with p - value < 0 . 05 . D . ( Left ) Venn diagram of the significant proteins shared by both IGR and WM cell lines . ( Right ) Histograms representing the metastatic and primitive signatures H / M ratios . F . Confocal fluorescence images of Proteostat ( 1 : 2000 , red spots ) and DAPI staining ( blue ) , scale bar is 10µm . G . Quantitation of aggregates / cell in IGRs cell lines by immunofluorescence analysis , left panel ; fluorescence gain of soluble proteins treated with Proteostat reagent , right panel . ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) .

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Figure 2B. Scatter plot of identified and quantified proteins in the secretome of primitive IGR39 and metastatic IGR37. Red dots represent proteins that were significant with FDR <0.05, blue dots represent proteins with p-value<0.05. C. Scatter plot of identified and quantified proteins fin the secretome of primitive WM115 and metastatic WM266.4. Red dots represent proteins that were significant with FDR <0.05, blue dots represent proteins with p-value<0.05. D. (Left)Venn diagram of the significant proteins shared by both IGR and WM cell lines. (Right) Histograms representing the metastatic and primitive signatures H/M ratios.F. Confocal fluorescence images of Proteostat (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. G. Quantitation of aggregates/cell in IGRs cell lines by immunofluorescence analysis, left panel; fluorescence gain of soluble proteins treated with Proteostat reagent, right panel. (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).


Figure 3A . Absolute quantitation of 24 , 25 and 27 - hydroxycholesterol in primitive and metastatic melanoma cells as indicated . ( T - test analysis , * * * p - value < 0 . 001 , N = 3 biological replicates , data are mean ± SD ) . B . Histogram representing R square of correlation analysis between absolute quantitation of 24 , 25 and 27 - hydroxycholesterol and label free quantitation of APOE in a cohort of primitive ( A375 , IPC298 ) and metastatic ( SKMEL5 , SKMEL28 ) melanoma cell lines . C . Confocal fluorescence images of anti - HMB45 PMEL antibody signal ( green ) and DAPI staining ( blue ) in IGR37 and IGR39 . Scale bar is 10µm . D . Western blot on IGR37 and 39 cellular lysates probed with anti - HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates .

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Figure 3A. Absolute quantitation of 24, 25 and 27-hydroxycholesterol in primitive and metastatic melanoma cells as indicated. (T-test analysis, ***p-value<0.001, N=3 biological replicates, data are mean ±SD).B. Histogram representing R square of correlation analysis between absolute quantitation of 24, 25 and 27-hydroxycholesterol and label free quantitation of APOE in a cohort of primitive (A375, IPC298) and metastatic (SKMEL5, SKMEL28) melanoma cell lines.C. Confocal fluorescence images of anti-HMB45 PMEL antibody signal (green) and DAPI staining (blue) in IGR37 and IGR39. Scale bar is 10µm.D. Western blot on IGR37 and 39 cellular lysates probed with anti-HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates.


Figure 4A - D . Immunofluorescence images with Proteostat ( red ) and DAPI ( blue ) staining on ( A ) human normal skin , ( B ) samples of primitive melanomas , ( C ) melanoma metastases in brain and ( D ) melanoma metastases in lung . E . Details of brain metastases . Scale Bar is 30µm . F . Quantitation of Proteostat positive dots in primitive vs metastatic melanoma tissues : 6 tissues from metastatic lesions and 6 from primitive melanoma lesions were analysed . For each tissue two sections were quantified . T - test analysis was applied . T - test analysis , * * = p - value < 0 . 01 ( N = 6 , data are mean ± SEM ) .

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Figure 4A-D. Immunofluorescence images with Proteostat (red) and DAPI (blue) staining on (A) human normal skin, (B) samples of primitive melanomas, (C) melanoma metastases in brain and (D) melanoma metastases in lung.E. Details of brain metastases. Scale Bar is 30µm. F. Quantitation of Proteostat positive dots in primitive vs metastatic melanoma tissues: 6 tissues from metastatic lesions and 6 from primitive melanoma lesions were analysed. For each tissue two sections were quantified. T-test analysis was applied. T-test analysis, ** = p-value<0.01 (N=6, data are mean ±SEM).


Figure 5A . Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB - 360 . B . Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB - 360 . D . Confocal fluorescence images of Proteostat signal ( 1 : 2000 , red spots ) and DAPI staining ( blue ) , scale bar is 10µm . E . Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software . ( T - test analysis , * * = p - value < 0 . 01 , N = 3 biological replicates , data are mean ± SD ) . F . Quantitation , by immunofluorescence analysis , of YAP in different cellular compartments ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) . Images were quantified by subdividing cells into mostly cytosolic YAP ( Cytosol ) , mostly nuclear YAP ( Nuclear ) , or equal distribution ( Total ) from three biological replicates . G . mRNA levels of CTGF measured by real - time PCR in IGR37 treated with DMSO or NB - 360 ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) . H . CTGF mRNA level measured by real - time PCR in IGR39 treated with IGR39 conditioned medium ( CTRL ) or with IGR37 conditioned medium ( CM ) , N = 4 biological replicates . T - test analysis , * * * p - value < 0 . 001 , data are mean ± SD . I . CTGF mRNA level measured by real - time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils ( 0 . 5µM ) , N = 3 biological replicates . T - test analysis : * * * p - value < 0 . 001 , data are mean ± SD . J . CTGF mRNA level measured by real - time PCR in IGR37 treated with DMSO , NB - 360 or NB - 360 plus recombinant PMEL amyloid fibrils ( 0 . 5µM ) , N = 3 biological replicates . T - test analysis * = p - value < 0 . 05 , * * = p - value < 0 . 01 . Data are mean ± SD .

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Figure 5A. Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB-360.B. Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB-360.D. Confocal fluorescence images of Proteostat signal (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. E. Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software. (T-test analysis, ** = p-value<0.01, N=3 biological replicates, data are mean ±SD). F. Quantitation, by immunofluorescence analysis, of YAP in different cellular compartments (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). Images were quantified by subdividing cells into mostly cytosolic YAP (Cytosol), mostly nuclear YAP (Nuclear), or equal distribution (Total) from three biological replicates.G. mRNA levels of CTGF measured by real-time PCR in IGR37 treated with DMSO or NB-360 (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).H. CTGF mRNA level measured by real-time PCR in IGR39 treated with IGR39 conditioned medium (CTRL) or with IGR37 conditioned medium (CM), N=4 biological replicates. T-test analysis, *** p-value <0.001, data are mean ±SD.I. CTGF mRNA level measured by real-time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis: *** p-value <0.001, data are mean ±SD.J. CTGF mRNA level measured by real-time PCR in IGR37 treated with DMSO, NB-360 or NB-360 plus recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis* = p-value<0.05, ** = p-value<0.01. Data are mean ±SD.