Figure 2B , C Kinetic change of mRNA expression of Top1 , Btbd1 , Tdp1 , and Srsf1 in microglia following LPS / IFNγ stimulation ( n = 6 , technical replicates ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using two - way ANOVA with Dunnett ' s Multiple Comparison test for ( B and C ) , D , E The protein expression of TOP1 was detected by Western blotting ( n = 3 , biological replicates ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test . F - H Immunostaining of TOP1 ( red ) in stimulated microglia ( 4 h after LPS / IFNγ stimulation ) or resting microglia , co - stained with Hoechst ( blue ) ( bar = 20 μm ) ; for the lower - left panel in ( H ) , n = 3 technical replicates per group ( 2 to 5 random fields per well were analyzed ) ; for the lower right panel , 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using the student ' s unpaired two - tailed t - test . G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti - F4 / 80 ( green ) , TOP1 ( magenta ) , and nuclei with Hoechst ( blue ) . For the left panels , bar = 400 μm ; for the magnified panels , bar = 100 μm . The yellow arrowhead indicates a representative F4 / 80 + cell with high TOP1 expression . I A magnified image of the selected region ( white box ) in ( G ) ; bar = 50 F4 / 80 + cell with increased TOP1 expression in the nucleus in ( G ) . J The mean fluorescent intensity of TOP1 expression in the white matter in ( G ) was graphed ; n = 3 to 4 mice per group with 3 - 5 sections from each mouse . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test . K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak ( scored 3 ) or control mice were examined by Western blotting . L The quantification of TOP1 protein expression of Western blot bands in ( K ) with normalization to GAPDH and β - actin ( n = 3 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test . M , Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) . TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test . O Microglia were sorted from LPS challenged mice ( i . p ) after 4 h or naive mice , and the mRNA expression of TOP1 was graphed ( n = 5 or 6 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test . P Brain sections from healthy control and MS brain tissues , including the active MS lesion , chronic active lesion , and the normal - appearing white matter ( NAWM ) , were stained with anti - HLA - DR , anti - TOP1 , and co - stained with Hoechst ( bar = 50 μm ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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

Figure 2B, C Kinetic change of mRNA expression of Top1, Btbd1, Tdp1, and Srsf1 in microglia following LPS/IFNγ stimulation (n = 6, technical replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using two-way ANOVA with Dunnett's Multiple Comparison test for (B and C),D, E The protein expression of TOP1 was detected by Western blotting (n = 3, biological replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.F-H Immunostaining of TOP1 (red) in stimulated microglia (4 h after LPS/IFNγ stimulation) or resting microglia, co-stained with Hoechst (blue) (bar = 20 μm); for the lower-left panel in (H), n = 3 technical replicates per group (2 to 5 random fields per well were analyzed); for the lower right panel, 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using the student's unpaired two-tailed t-test.G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti-F4/80 (green), TOP1 (magenta), and nuclei with Hoechst (blue). For the left panels, bar = 400 μm; for the magnified panels, bar = 100 μm. The yellow arrowhead indicates a representative F4/80+ cell with high TOP1 expression.I A magnified image of the selected region (white box) in (G); bar = 50 F4/80+ cell with increased TOP1 expression in the nucleus in (G). J The mean fluorescent intensity of TOP1 expression in the white matter in (G) was graphed; n = 3 to 4 mice per group with 3-5 sections from each mouse. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak (scored 3) or control mice were examined by Western blotting. L The quantification of TOP1 protein expression of Western blot bands in (K) with normalization to GAPDH and β-actin (n = 3 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.M, Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples).TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.O Microglia were sorted from LPS challenged mice (i.p) after 4 h or naive mice, and the mRNA expression of TOP1 was graphed (n = 5 or 6 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.P Brain sections from healthy control and MS brain tissues, including the active MS lesion, chronic active lesion, and the normal-appearing white matter (NAWM), were stained with anti-HLA-DR, anti-TOP1, and co-stained with Hoechst (bar = 50 μm).


Figure 3C , D Immunostaining of iNOS ( red ) and Phalloidin ( green ) in microglia ( n = 3 technical replicates with more than 5 random fields per well ) ( bar = 50 μm ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses were performed using one - way ANOVA with Dunnett ' s Multiple Comparison Test for ( D )

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

Figure 3C, D Immunostaining of iNOS (red) and Phalloidin (green) in microglia (n = 3 technical replicates with more than 5 random fields per well) (bar = 50 μm). Data information: Data shown are mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; n.s. = no statistical significance. Statistical analyses were performed using one-way ANOVA with Dunnett's Multiple Comparison Test for (D)


Figure 4B Microglia in the hippocampus and thalamus were stained with Iba1 ( red ) ; bar = 50 μm ; microglia in the yellow boxes were presented in ( E ) . C , D Fluorescent intensity of Iba1 in hippocampus and thalamus in ( B ) ; n = 2 , 4 , 5 mice for naive , LPS , and LPS + TPT group , respectively ( 2 - 6 sections per mouse were analyzed ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses comparing the LPS _ PBS group with the LPS _ TPT group were performed using the Student ' s unpaired two - tailed t - testL , M Cytokine array on brain homogenates after 24 h of LPS challenge ( n = 2 mice for the naïve group ; n = 3 mice for the LPS and LPS + TPT groups ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4B Microglia in the hippocampus and thalamus were stained with Iba1 (red); bar = 50 μm; microglia in the yellow boxes were presented in (E).C, D Fluorescent intensity of Iba1 in hippocampus and thalamus in (B); n = 2, 4, 5 mice for naive, LPS, and LPS + TPT group, respectively (2-6 sections per mouse were analyzed). Data information: Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses comparing the LPS_PBS group with the LPS_TPT group were performed using the Student's unpaired two-tailed t-testL, M Cytokine array on brain homogenates after 24 h of LPS challenge (n = 2 mice for the naïve group; n = 3 mice for the LPS and LPS + TPT groups).


Figure 6K Spinal cord sections dissected from Day 30 from the EAE batch in ( H ) were stained with CD11b ( red ) , Fluoromyelin ( green ) , and Hoechst ( blue ) ( bar = 200 μm ) . L The CD11b immunofluorescent intensity in ( K ) was graphed ( n = 2 mice for the control group ; n = 4 mice for the EAE - PBS and EAE - TPT groups ) . M The percentage of demyelinated area in the white matter of the spinal cord was graphed ( n = 2 mice for the control group , n = 4 mice for the EAE - PBS and EAE - TPT groups ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; the Student ' s unpaired two - tailed t - test was used for comparing the EAE - PBS group with the EAE - TPT group in L , and M .

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

Figure 6K Spinal cord sections dissected from Day 30 from the EAE batch in (H) were stained with CD11b (red), Fluoromyelin (green), and Hoechst (blue) (bar = 200 μm). L The CD11b immunofluorescent intensity in (K) was graphed (n = 2 mice for the control group; n = 4 mice for the EAE-PBS and EAE-TPT groups). M The percentage of demyelinated area in the white matter of the spinal cord was graphed (n = 2 mice for the control group, n = 4 mice for the EAE-PBS and EAE-TPT groups). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; the Student's unpaired two-tailed t-test was used for comparing the EAE-PBS group with the EAE-TPT group in L, and M.


Figure 1A RNA - seq . of freshly isolated , proliferating and differentiating satellite cells reveals an increased expression of linc - MYH in proliferating and differentiating MuSCs , but no expression is found in in freshly isolated MuSCs . Expression of linc - MYH is confined to skeletal muscle in mouse ( B ) tissues . Expression of linc - MYH is confined to skeletal muscle in human ( C ) tissues . D Expression of linc - MYH is not detected in limb buds at E10 . 5 ( n = 3 ) , E13 . 5 ( n = 3 ) and E15 . 5 ( n = 3 ) . Expression of linc - MYH in hindlimb muscle of E18 . 5 , newborn and 7 day old animals ( n = 3 / 3 / 3 ) . Expression of linc - MYH in m . soleus ( soleus ) , m . tibialis anterior ( TA ) and and m . extensor digitorum longus ( EDL ) muscle at 3 weeks ( n = 3 ) and 8 weeks of age ( n = 5 ) . Biological replicates were used for all PCR experiments , data are mean ± S . E . M . E Murine linc - Myh is localized in nuclear extracts of C2C12 cells . Xist ( nuclear ) and Gapdh ( mainly cytoplasmic ) were used as controls ( n = 2 nuclear / 3 cytoplasmic biological replicates . ) Data are mean ± S . E . M . F - H RNA - FISH identifies linc - Myh in nuclei ( DAPI , blue ) of C2C12 myoblasts . ( G ) Adipor ( cytoplasmic ) and ( H ) Xist ( nuclear ) were used as controls . ( J ) Heat map of proteins pulled down in the same experiments as in I , demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc - MYH . Blue color indicates pull - down of the respective protein , white color indicates that the protein was not detected in the respective sample . RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from murine C2C12 cells ( K ) INO80 in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR . RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from human HSMM cells ( L ) . INO80 II in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

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 B-PANEL_START 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 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 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 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 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 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

Figure 1A RNA-seq. of freshly isolated, proliferating and differentiating satellite cells reveals an increased expression of linc-MYH in proliferating and differentiating MuSCs, but no expression is found in in freshly isolated MuSCs.Expression of linc-MYH is confined to skeletal muscle in mouse (B) tissues.Expression of linc-MYH is confined to skeletal muscle in human (C) tissues.D Expression of linc-MYH is not detected in limb buds at E10.5 (n = 3), E13.5 (n = 3) and E15.5 (n = 3). Expression of linc-MYH in hindlimb muscle of E18.5, newborn and 7 day old animals (n = 3/3/3). Expression of linc-MYH in m. soleus (soleus), m. tibialis anterior (TA) and and m. extensor digitorum longus (EDL) muscle at 3 weeks (n = 3) and 8 weeks of age (n = 5). Biological replicates were used for all PCR experiments, data are mean ± S.E.M.E Murine linc-Myh is localized in nuclear extracts of C2C12 cells. Xist (nuclear) and Gapdh (mainly cytoplasmic) were used as controls (n = 2 nuclear / 3 cytoplasmic biological replicates.) Data are mean ± S.E.M.F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.(J) Heat map of proteins pulled down in the same experiments as in I, demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc-MYH. Blue color indicates pull-down of the respective protein, white color indicates that the protein was not detected in the respective sample.RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from murine C2C12 cells (K) INO80 in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from human HSMM cells (L). INO80 II in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


Figure 2Body weight ( A ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 24 KO / 17 WT ; Student ' s t test , two - tailed , * * p < 0 . 01 ) tibia length ( B ) of male linc - MYH KO and ctrl mice of 10 weeks ( n = 9 KO / 13 WT ; Student ' s t test , two - tailed , not significant ) . Data are mean ± S . E . M . C , D Weight of m . tibialis anterior ( TA ) and m . digitorum longus ( EDL ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 9 KO / 10 WT for TA , n = 10 KO / 11 WT for EDL ; Student ' s t test , two - tailed , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data are mean ± S . E . M . Size distribution of fibers in cross sections of TA ( E , F ) and EDL ( G , H ) muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M . Size distribution of fibers in cross sections of TA and EDL muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M . K Numbers of myonuclei in cross sections of TA and EDL muscle ( TA : n = 6 KO / 6 WT , EDL n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed , * * p < 0 . 01 , * p = 0 . 05 ) Data are mean ± S . E . M . L - O Number of myonuclei / fiber and of Pax7pos ( red ) MuSCs ( red arrows ) on myofibers isolated from flexor digitorum brevis muscle . Nuclei were stained using DAPI ( blue ) . Scale bar in L indicates 50 µm for L and M . ( N ) The number of myonuclei / isolated fiber is increased in linc - MYH KO compared to WT myofibers ( n = 4 KO / 4 WT , > 26 fibers / animal ; Mann - Whitney test two - tailed , * p < 0 . 05 ) . ( O ) Number of MuSCs on isolated fibers from linc - MYH KO and WT mice ( n = 4 KO / 4 WT animals , Mann - Whitney test two - tailed ; * * * * p < 0 . 0001 ) . Data are mean ± S . E . M . P - S Number of Pax7 ( red ) positive MuSCs in cross sections of tibialis anterior muscle ( P - R ) ; the relative amount of quiescent MuSCs identified by double staining ( yellow arrows ) for Pax7 and CalcR ( green ) does not significantly differ between WT and linc - MYH KO animals ( S ) . Nuclei were stained with DAPI ( blue ) . Scale bar in P indicates 25 µm for P and Q . ( n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed ; * p = 0 . 05 , ns : not significant ) . All data are mean ± S . E . M . T The number of MuSC - related events in FACS experiments is significantly increased in linc - MYH - KO animals ( n = 7 KO / 7 WT , Student ' s t test , two - tailed , * p = 0 . 0126 ) . Data are mean ± S . E . M . U - V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc - MYH KO ( V ) compared to WT mice ( U ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 2Body weight (A) of male linc-MYH KO and ctrl mice at 10 weeks (n = 24 KO/17 WT; Student's t test, two-tailed, **p < 0.01)tibia length (B) of male linc-MYH KO and ctrl mice of 10 weeks (n = 9 KO/13 WT; Student's t test, two-tailed, not significant). Data are mean ± S.E.M.C, D Weight of m. tibialis anterior (TA) and m. digitorum longus (EDL) of male linc-MYH KO and ctrl mice at 10 weeks (n = 9 KO/10 WT for TA, n = 10 KO/11 WT for EDL; Student's t test, two-tailed, **p < 0.01, ***p<0.001). Data are mean ± S.E.M.Size distribution of fibers in cross sections of TA (E, F) and EDL (G, H) muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.Size distribution of fibers in cross sections of TA and EDL muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.K Numbers of myonuclei in cross sections of TA and EDL muscle (TA: n = 6 KO/6 WT, EDL n = 3 KO/3 WT animals, Mann-Whitney test one-tailed, **p<0.01, *p = 0.05) Data are mean ± S.E.M.L-O Number of myonuclei / fiber and of Pax7pos (red) MuSCs (red arrows) on myofibers isolated from flexor digitorum brevis muscle. Nuclei were stained using DAPI (blue). Scale bar in L indicates 50 µm for L and M. (N) The number of myonuclei / isolated fiber is increased in linc-MYH KO compared to WT myofibers (n = 4 KO/4 WT, > 26 fibers/animal; Mann-Whitney test two-tailed, * p < 0.05). (O) Number of MuSCs on isolated fibers from linc-MYH KO and WT mice (n = 4 KO/4 WT animals, Mann-Whitney test two-tailed; ****p < 0.0001). Data are mean ± S.E.M.P-S Number of Pax7 (red) positive MuSCs in cross sections of tibialis anterior muscle (P-R); the relative amount of quiescent MuSCs identified by double staining (yellow arrows) for Pax7 and CalcR (green) does not significantly differ between WT and linc-MYH KO animals (S). Nuclei were stained with DAPI (blue). Scale bar in P indicates 25 µm for P and Q. (n = 3 KO/3 WT animals, Mann-Whitney test one-tailed; *p = 0.05, ns: not significant). All data are mean ± S.E.M.T The number of MuSC-related events in FACS experiments is significantly increased in linc-MYH-KO animals (n = 7 KO/7 WT, Student's t test, two-tailed, *p = 0.0126). Data are mean ± S.E.M.U-V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc-MYH KO (V) compared to WT mice (U).


Figure 3RNA in situ hybridization proximity ligation assay ( rISH - PLA ) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al . . ( B - C ) rISH - PLA confirms the proximity of linc - MYH to endogenous INO80 - V5 in the nucleus of proliferating WT MuSCs ( B ) . The specific signal is lost in linc - MYH deficient MuSCs ( C ) . ( n = 3 WT / 3 KO biological replicates ; Mann - Whitney test , * p = 0 . 05 ) . Data are mean ± S . E . M . E - I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 ( green ) and EdU incorporation ( red ) . MuSCs without ( green arrows ) and with EdU - labeling ( red arrows ) are indicated . ( E , F ) Deletion of linc - MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU - positive MuSCs . ( G - I ) The increase in both the number of MuSCs and EdU positive MuSCs in linc - MYH mutant muscle is abolished by constitutive ( G : Pax7 - Crepos / INO80 - / - , Ino80 KO ; H : linc - MYH - / - / Pax7 - Crepos / INO80 - / - , dKO ) and induced ( I : linc - MYH - / - / Pax7 - CreERT2pos / INO80 - / - , dKO CreERT ) deletion of INO80 in Pax7 expressing cells . Statistical evaluation of the number of muscle stem cells observed in TA muscles ( J ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) . Statistical evaluation of the ratio of EdU - incorporating MuSCs relative to all Pax7 - positive MuSCs ( K ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) . Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes ( L ) ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 3RNA in situ hybridization proximity ligation assay (rISH-PLA) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al.. (B-C) rISH-PLA confirms the proximity of linc-MYH to endogenous INO80-V5 in the nucleus of proliferating WT MuSCs (B). The specific signal is lost in linc-MYH deficient MuSCs (C). (n = 3 WT/3 KO biological replicates; Mann-Whitney test, *p = 0.05). Data are mean ± S.E.M.E-I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 (green) and EdU incorporation (red). MuSCs without (green arrows) and with EdU-labeling (red arrows) are indicated. (E, F) Deletion of linc-MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU-positive MuSCs. (G-I) The increase in both the number of MuSCs and EdU positive MuSCs in linc-MYH mutant muscle is abolished by constitutive (G: Pax7-Crepos / INO80-/-, Ino80 KO; H: linc-MYH-/- / Pax7-Crepos / INO80-/-, dKO) and induced (I: linc-MYH-/- / Pax7-CreERT2pos / INO80-/-, dKO CreERT) deletion of INO80 in Pax7 expressing cells.Statistical evaluation of the number of muscle stem cells observed in TA muscles (J). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).Statistical evaluation of the ratio of EdU-incorporating MuSCs relative to all Pax7-positive MuSCs (K). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes (L) (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


Figure 4A - D Isolated linc - MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates . Deletion of INO80 eliminates the difference between Pax7 - Crepos / INO80 - / - ( Ino80 KO ) and linc - MYH - / - / linc - MYH - / - / Pax7 - Crepos / INO80 - / - ( dKO ) . Images are representative images for cultures after 13 days in culture . E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs . Cell confluence was monitored by a live cell imaging analysis system for 13 days ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs ) . F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs , Student ' s t test , two - tailed ; * p < 0 . 05 , ns = not significant ) . Data are mean ± S . E . M . G - I Gene set enrichment analysis ( GSEA , broadinstitute . org ) of microarray data from proliferating MuSCs . Comparison of linc - MYH - / - vs . WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation ( G ) , enrichment of genes containing cis regulatory elements for YY1 ( H ) and p53 ( I ) . Enrichments are highly significant using the most conservative familywise - error rate method ( FWER ) . Enrichments were lost in linc - MYH - / - MuSCs after additional deletion of INO80 ( dKOs ) compared to Pax7 - Crepos / INO80 - / - MuSCs . Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G - I in linc - MYH - / - vs . WT MuSCs and in linc - MYH - / - / Pax7 - Crepos / INO80 - / - dKO vs . Pax7 - Crepos / INO80 - / - MuSCs . Expression of up - regulated genes in linc - MYH - / - was normalized after additional deletion of INO80 ( dKOs ) , indicating that effects of linc - MYH completely depend on INO80 ( WT n = 4 , linc - MYH - / - n = 4 , Pax7 - Crepos / INO80 - / - n = 4 , Pax7 - Cre dKO n = 3 animals used for independent isolations of MuSCs ) . Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G - I in linc - MYH - / - vs . WT MuSCs and in linc - MYH - / - / Pax7 - Crepos / INO80 - / - dKO vs . Pax7 - Crepos / INO80 - / - MuSCs . Expression of up - regulated genes in linc - MYH - / - was normalized after additional deletion of INO80 ( dKOs ) , indicating that effects of linc - MYH completely depend on INO80 ( WT n = 4 , linc - MYH - / - n = 4 , Pax7 - Crepos / INO80 - / - n = 4 , Pax7 - Cre dKO n = 3 animals used for independent isolations of MuSCs ) . Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G - I in linc - MYH - / - vs . WT MuSCs and in linc - MYH - / - / Pax7 - Crepos / INO80 - / - dKO vs . Pax7 - Crepos / INO80 - / - MuSCs . Expression of up - regulated genes in linc - MYH - / - was normalized after additional deletion of INO80 ( dKOs ) , indicating that effects of linc - MYH completely depend on INO80 ( WT n = 4 , linc - MYH - / - n = 4 , Pax7 - Crepos / INO80 - / - n = 4 , Pax7 - Cre dKO n = 3 animals used for independent isolations of MuSCs ) .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 4A-D Isolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates. Deletion of INO80 eliminates the difference between Pax7-Crepos / INO80-/- (Ino80 KO) and linc-MYH-/- / linc-MYH-/-/ Pax7-Crepos / INO80-/- (dKO). Images are representative images for cultures after 13 days in culture.E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs. Cell confluence was monitored by a live cell imaging analysis system for 13 days (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs).F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs, Student's t test, two-tailed; *p < 0.05, ns = not significant). Data are mean ± S.E.M.G-I Gene set enrichment analysis (GSEA, broadinstitute.org) of microarray data from proliferating MuSCs. Comparison of linc-MYH-/- vs. WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation (G), enrichment of genes containing cis regulatory elements for YY1 (H) and p53 (I). Enrichments are highly significant using the most conservative familywise-error rate method (FWER). Enrichments were lost in linc-MYH-/- MuSCs after additional deletion of INO80 (dKOs) compared to Pax7-Crepos / INO80-/- MuSCs.Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).


Figure 5A - D γ - H2AX staining of proliferating MuSCs from WT ( A ) , linc - MYH deficient ( B ) , Pax7 - Crepos / INO80 KO ( C ) , and linc - MYH / INO80 dKO ( D ) mice . Deletion of INO80 results in increased γ - H2AX signals while deletion of linc - MYH has no effects ( asterisk : low γ - H2AX signals , arrow : γ - H2AX - foci detected in nuclei , arrowhead : pan - nuclear γ - H2AX signal . E - G Statistical evaluation of the presence of γ - H2AX signals in MuSCs of different genotypes ( n = 3 / 3 / 3 / 3 animals used for independent isolations of MuSCs , two technical replicates for each animal and at least 164 cells were counted , Mann - Whitney test , * * p < 0 . 01 . Data are mean ± s . e . m . ) . H - I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery ( H ) and enrichment of genes involved in chromosome maintenance ( I ) in WT compared to Pax7 - Crepos INO80 - / - MuSCs . Enrichment of these or similar gene sets was not observed when comparing WT to linc - MYH - / - MuSCs .

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Figure 5A-D γ-H2AX staining of proliferating MuSCs from WT (A), linc-MYH deficient (B), Pax7-Crepos / INO80 KO (C), and linc-MYH/INO80 dKO (D) mice. Deletion of INO80 results in increased γ-H2AX signals while deletion of linc-MYH has no effects (asterisk: low γ-H2AX signals, arrow: γ-H2AX-foci detected in nuclei, arrowhead: pan-nuclear γ-H2AX signal.E-G Statistical evaluation of the presence of γ-H2AX signals in MuSCs of different genotypes (n = 3/3/3/3 animals used for independent isolations of MuSCs, two technical replicates for each animal and at least 164 cells were counted, Mann-Whitney test, **p<0.01. Data are mean ± s.e.m.).H-I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery (H) and enrichment of genes involved in chromosome maintenance (I) in WT compared to Pax7-Crepos INO80-/- MuSCs. Enrichment of these or similar gene sets was not observed when comparing WT to linc-MYH-/- MuSCs.


Figure 6A , B Co - Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc - MYH KO mice followed by Western Blot analysis . ( B ) Statistical analysis of Co - IP / Western Blot data . The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc - MYH , while the amounts of YY1 , WDR5 and TFPT pulled down by INO80 increase ( n = 3 WT / 3 linc - MYH KO animals ; one - way ANOVA , multiple comparisons by Fishers LSD test , * p < 0 . 05 ) . Data are mean ± S . E . M . C - K Proximity ligation assays between INO80 and YY1 ( C , D ) , INO80 and WDR5 ( F , G ) , and INO80 and RuvBl2 ( I , J ) in proliferating MuSCs . Red signals indicate close proximity of INO80 - V5 and the respective interacting protein . Nuclei were stained using DAPI ( blue ) . Statistical evaluation of proximity ligation assays indicating increased interactions of INO80 - V5 and YY1 ( E , n = 3 / 3 animals , 3 independent wells per animal , two - tailed students t - test , * p < 0 . 05 ) and of INO80 - V5 to WDR5 ( H , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , * * p < 0 . 01 ) . No increase in proximity was detected between Ino80 - V5 and Ruvbl2 ( K , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , ns : not significant ) . Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays . No signals were detected for these controls . Data are mean ± S . E . M .

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Figure 6A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis. (B) Statistical analysis of Co-IP/Western Blot data. The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc-MYH, while the amounts of YY1, WDR5 and TFPT pulled down by INO80 increase (n = 3 WT / 3 linc-MYH KO animals; one-way ANOVA, multiple comparisons by Fishers LSD test, *p<0.05). Data are mean ± S.E.M.C-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.


Figure 1 ( A ) Coomassie stained SDS - PAGE showing purified T . thermophila tubulin . ( B ) A representative cryo - electron micrograph of paclitaxel - stabilized T . thermophila MTs . ( C ) Kymographs obtained by TIRF microscopy showing the dynamics of T . thermophila MTs at the indicated concentrations in µM .

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Figure 1(A) Coomassie stained SDS-PAGE showing purified T. thermophila tubulin.(B) A representative cryo-electron micrograph of paclitaxel-stabilized T. thermophila MTs.(C) Kymographs obtained by TIRF microscopy showing the dynamics of T. thermophila MTs at the indicated concentrations in µM.


Figure 2 ( D ) Plot showing the time course of T . thermophila cell growth in the presence of CA4 ( 20µM ) or parabulin ( 50µM ; error bars corresponding to the standard deviation of triplicate biological measurements ) . Control experiments are done with DMSO alone .

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Figure 2(D) Plot showing the time course of T. thermophila cell growth in the presence of CA4 (20µM) or parabulin (50µM; error bars corresponding to the standard deviation of triplicate biological measurements). Control experiments are done with DMSO alone.


Figure 3 ( A ) Plot showing quantification of the T . thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs . Error bars represent the mean + / - 95 % CI . For each concentration , data is obtained from at least 2 independent experiments . For growth rates and growth lengths , n = 41 ( DMSO ) , n = 4 ( 0 . 04uM parabulin ) , n = 34 ( 0 . 04 uM colchicine ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison test ; DMSO vs parabulin P < 0001 ( * * * * ) ; DMSO vs colchicine p = 0 . 40 ( n . s . , not significant ) . ( B ) T . thermophila MT growth rates in the presence of the indicated drugs ( 40 μM ) . Error bars represent the mean + / - 95 % CI ( n = 4 ) . Rates were compared using one - way ANOVA with Tukey ' s multiple comparison test ; DMSO vs parabulin P = 0 . 25 ; DMSO vs colchicine P = 0 . 20 ( n . s . , not significant ) . ( D ) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt - MTs .

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Figure 3(A) Plot showing quantification of the T. thermophila MT growth events in TIRF microscopy MT dynamics reconstitution assays in the presence of the indicated drugs. Error bars represent the mean +/- 95% CI. For each concentration, data is obtained from at least 2 independent experiments. For growth rates and growth lengths, n=41(DMSO), n= 4 (0.04uM parabulin), n= 34 (0.04 uM colchicine). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P<0001 (****); DMSO vs colchicine p = 0.40 (n.s., not significant). (B) T. thermophila MT growth rates in the presence of the indicated drugs (40 μM). Error bars represent the mean +/- 95% CI (n=4). Rates were compared using one-way ANOVA with Tukey's multiple comparison test; DMSO vs parabulin P = 0.25; DMSO vs colchicine P = 0.20 (n.s., not significant).(D) Representative kymographs obtained by TIRF microscopy showing the effect of parabulin and colchicine on dynamic Tt-MTs.


Figure 4 ( A ) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample . The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100 % growth . Sub - confluent human fibroblasts were used to visualize potential compound effects during proliferation . The results represent the average of 9 readings ( 3 biological replicates with 3 technical replicates of each treatment ) ± standard error of the mean . ( B ) Replication of GFP - expressing T . gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging . Since vacuoles containing more than eight tachyzoites are not always easy to score , we tallied vacuoles above eight as being 8 + in size . At 36 hours , both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites ( three or more doublings ) . Vacuoles in parabulin - treated cultures contain significantly fewer parasites , indicating that replication is slowed . The graph shows three biological replicates with values normalized to 100 % for each condition and each replicate , error bars represent standard error of the mean between experiments . ( C ) The parabulin EC50 value for T . gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions . Error bars represent standard error of the mean . The results represent the average of 18 readings ( 6 biological replicates with 3 technical replicates of each treatment ) ± standard error of the mean . ( D ) Phase contrast of extracellular parasite cultures . Serial passage of T . gondii in 40 - 80 μM parabulin induces accumulation of aberrant tachyzoite forms . These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding .

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Figure 4(A) Evaluation of vertebrate cell toxicity using the MTT assay which detects metabolic activity proportionate to the number of viable cells in a sample. The absorbance measurements for all treatment conditions were normalized to the null control value which is set to 100% growth. Sub-confluent human fibroblasts were used to visualize potential compound effects during proliferation. The results represent the average of 9 readings (3 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.(B) Replication of GFP-expressing T. gondii tachyzoites in individual intracellular vacuoles can be followed by live cell imaging. Since vacuoles containing more than eight tachyzoites are not always easy to score, we tallied vacuoles above eight as being 8+ in size. At 36 hours, both null and vehicle control samples show most vacuoles harbor eight or more tachyzoites (three or more doublings). Vacuoles in parabulin-treated cultures contain significantly fewer parasites, indicating that replication is slowed. The graph shows three biological replicates with values normalized to 100% for each condition and each replicate, error bars represent standard error of the mean between experiments.(C) The parabulin EC50 value for T. gondii growth was determined using a plaque assay to measure a reduction in plaque area relative to control conditions. Error bars represent standard error of the mean. The results represent the average of 18 readings (6 biological replicates with 3 technical replicates of each treatment) ± standard error of the mean.(D) Phase contrast of extracellular parasite cultures. Serial passage of T. gondii in 40-80 μM parabulin induces accumulation of aberrant tachyzoite forms. These arise with MT defects that disconnect coordinated nuclear division and daughter cell budding.


Figure 2A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 24 , 48 and 72 hours and imaged after DNA staining with Hoechst . Green arrowheads indicate examples of prometaphase arrested cells , and the red arrowheads indicate examples of dead cells due to Plk1 downregulation . Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments . Scale bar , 20 μm . B ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours and imaged after DNA staining with Hoechst . C ) Quantification of nuclear size measurements from Fig 2B . Data derived from 3 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov test . D ) Solid phase transfection of nontargeting ( scrambled ) , GOLGA2 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Golga2 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . E ) Quantification of experiments in Fig 2D . Data derived from 3 independent experiments . P value ( scrambled vs GOLGA2 ) < 2e - 16 , Mann - Whitney U test . F ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Ki67 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . G ) Quantification of experiments in Fig 2F . Data derived from 3 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U test . H ) Comparison of phenotypic penetrance with liquid and solid phase transfection . RPE - 1 , HEK293T , NCI - H358 and NCI - N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers ( 2250 or 20000 cells / well ) . 5 days post transfection cell viability were measured by CellTiter - Glo . Boxplots represent values from at least 3 independent experiments containing 3 technical replicates . I ) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines . Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days . The raw values are background subtracted and normalized to the mock controls . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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 O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 2A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 24, 48 and 72 hours and imaged after DNA staining with Hoechst. Green arrowheads indicate examples of prometaphase arrested cells, and the red arrowheads indicate examples of dead cells due to Plk1 downregulation. Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments. Scale bar, 20 μm.B) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. C) Quantification of nuclear size measurements from Fig 2B. Data derived from 3 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.D) Solid phase transfection of nontargeting (scrambled), GOLGA2 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Golga2 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. E) Quantification of experiments in Fig 2D. Data derived from 3 independent experiments. P value (scrambled vs GOLGA2) < 2e-16, Mann-Whitney U test. F) Solid phase transfection of nontargeting (scrambled), MKI67 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. G) Quantification of experiments in Fig 2F. Data derived from 3 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test. H) Comparison of phenotypic penetrance with liquid and solid phase transfection. RPE-1, HEK293T, NCI-H358 and NCI-N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers (2250 or 20000 cells/well). 5 days post transfection cell viability were measured by CellTiter-Glo. Boxplots represent values from at least 3 independent experiments containing 3 technical replicates.I) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines. Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days. The raw values are background subtracted and normalized to the mock controls. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


Figure 3A ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting RNP complexes into WT RPE - 1TP53 - / - cells . Cells were fixed after 72 hours and imaged after DNA staining with Hoechst . Scale bar , 50μm . B ) Quantification of experiments in Fig 3A . Data derived from 2 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov test . C ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting RNP complexes into WT RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Ki67 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . D ) Quantification of experiments in Fig 3C . Data derived from 2 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U test . E ) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , cell viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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Figure 3A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. Scale bar, 50μm. B) Quantification of experiments in Fig 3A. Data derived from 2 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test. C) Solid phase transfection of nontargeting (scrambled), MKI67 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. D) Quantification of experiments in Fig 3C. Data derived from 2 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test. E) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


Figure 4A ) Viability of NCI - N87 and NCI - H358 cells upon transfection of 45 different gRNAs by solid phase transfection . Cas9 expressing NCI - N87 and NCI - H358 cell were seeded on pre - coated plates . 5 days post transfection , cell viability in each well were measured by CellTiter - Glo . Values were background subtracted , normalized to scrambled controls and plotted against each other . Blue dots represent scrambled controls , whereas the yellow dots represent POLR2A gRNAs . Green dots represent the genes that affect the viability in a cell line dependent manner . Results are representative of 3 independent experiments . B , C ) Logarithm of the odds ( LOD ) scores of cancer associated gene KOs in NCI - N87 and NCI - H358 cells derived from screens with gRNAs ( B ) and RNPs ( C ) . Bottom panels show distribution of positive ( POLR2A ) and negative ( scrambled ) controls for each experiment . LOD score of 3 is highlighted with a dashed line , indicating statistical significance .

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Figure 4A) Viability of NCI-N87 and NCI-H358 cells upon transfection of 45 different gRNAs by solid phase transfection. Cas9 expressing NCI-N87 and NCI-H358 cell were seeded on pre-coated plates. 5 days post transfection, cell viability in each well were measured by CellTiter-Glo. Values were background subtracted, normalized to scrambled controls and plotted against each other. Blue dots represent scrambled controls, whereas the yellow dots represent POLR2A gRNAs. Green dots represent the genes that affect the viability in a cell line dependent manner. Results are representative of 3 independent experiments.B, C) Logarithm of the odds (LOD) scores of cancer associated gene KOs in NCI-N87 and NCI-H358 cells derived from screens with gRNAs (B) and RNPs (C). Bottom panels show distribution of positive (POLR2A) and negative (scrambled) controls for each experiment. LOD score of 3 is highlighted with a dashed line, indicating statistical significance.


Figure 5A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting RNP complexes into human primary lung fibroblasts ( HLF - 1 ) , primary lung adenocarcinoma cells ( LAD - 1 ) or RPE - 1TP53 - / - cells . 72 hours post transfection cells were stained with Hoechst and imaged . Boxplots represent values from 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs Plk1 ) < 0 . 05 . In the right panels , representative images for each cell line is shown after RNP transfection targeting PLK1 . Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation . Scale bars indicate 20μm . B ) Two human primary lung fibroblasts ( HLF ) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma ( LSC ) or lung adenocarcinoma ( LAD ) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , cell viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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 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

Figure 5A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting RNP complexes into human primary lung fibroblasts (HLF-1), primary lung adenocarcinoma cells (LAD-1) or RPE-1TP53 -/- cells. 72 hours post transfection cells were stained with Hoechst and imaged. Boxplots represent values from 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs Plk1) < 0.05. In the right panels, representative images for each cell line is shown after RNP transfection targeting PLK1. Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation. Scale bars indicate 20μm.B) Two human primary lung fibroblasts (HLF) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma (LSC) or lung adenocarcinoma (LAD) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


Figure 1 ( C ) Mass spectra of recombinant HTTex1 23Q after 16 hour of co - incubation with by TBK1 showing complete phosphorylation of S13 and S16 . The lower panel is a representative western blot of the same phosphorylation reaction ( upper panel ) using anti - pT3 ( CHDI - 90001528 - 1 ) , pS13 HTT ( CHDI - 90001039 - 1 ) and pS16 ( ZCH11020 - generated in house ) , antibodies ( ab ) after the indicated phosphorylation reaction times . ( D ) Representative western blot of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q and 72Q eGFP with the indicated kinases for 48 hour in HEK293T cells . ( E ) Quantification of the fold changes in the HTT pS13 and pT3 ratios to total HTT compared to empty vector ( EV ) control upon coexpression of the indicated kinase from the experiments like in D .

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Figure 1(C) Mass spectra of recombinant HTTex1 23Q after 16 hour of co-incubation with by TBK1 showing complete phosphorylation of S13 and S16. The lower panel is a representative western blot of the same phosphorylation reaction (upper panel) using anti-pT3 (CHDI-90001528-1), pS13 HTT (CHDI-90001039-1) and pS16 (ZCH11020 -generated in house), antibodies (ab) after the indicated phosphorylation reaction times.(D) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q and 72Q eGFP with the indicated kinases for 48 hour in HEK293T cells.(E) Quantification of the fold changes in the HTT pS13 and pT3 ratios to total HTT compared to empty vector (EV) control upon coexpression of the indicated kinase from the experiments like in D.


Figure 2 ( A ) Representative western blot of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q eGFP , mutants with S to A substitutions at T3 or S13 / S16 or HTTex1 72Q eGFP with TBK1 or TBK1 kinase - dead ( KD ) for 48 hour in HEK293T cells ( the additional high molecular weight HTTex1 and HTT pS13 band is possibly due to the co - occurrence of phosphorylation at both S13 and T3 ) . ( B ) Quantification of the fold changes in the HTT pS13 ( upper panel ) and pT3 ( lower panel ) ratio to total HTT compared to those of the kinase - dead mutant from the experiments like in A ( n = 3 ) . ( C ) Representative western blot of HTT ( ab - MAB2166 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells . ( D ) Quantification of HTT pS13 using the Singulex assay ( using ab - MW1 as capture and CHDI - 90001039 or 2B7 as detection antibodies for pS13 or total HTT ) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells from the experiment in C ( representative of 3 repeats ) . ( E ) Representative western blot of HTT ( ab - MAB2166 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) upon coexpression of HTT - FL 48Q or HTT - FL 48Q S13A / S16A with the indicated kinase for 24 hour in HEK293T cells . ( F ) Quantification of HTT pS13 using the Singulex assay ( using ab - MW1 as capture and CHDI - 90001039 or 2B7 as detection antibodies for pS13 or total HTT ) upon coexpression of HTT - FL 48Q or HTT - FL 48Q S13A / S16A with the indicated kinase for 24 hour in HEK293T cells from the experiments likein E ( n = 3 ) . ( G ) Representative western blot of immunoprecipitated ( IP ) HTT ( ab - MAB2166 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) after lentivirus ( LV ) - mediated overexpression of TBK1 or TBK1 KD for 72 hour in rat primary striatal neuronal cells . ( H ) Quantification of the fold changes in the HTT pS13 ratio to total HTT compared to those of the kinase - dead mutant from the experiments like in G ( n = 3 ) .

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 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 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 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

Figure 2(A) Representative western blot of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP, mutants with S to A substitutions at T3 or S13/S16 or HTTex1 72Q eGFP with TBK1 or TBK1 kinase-dead (KD) for 48 hour in HEK293T cells (the additional high molecular weight HTTex1 and HTT pS13 band is possibly due to the co-occurrence of phosphorylation at both S13 and T3). (B) Quantification of the fold changes in the HTT pS13 (upper panel) and pT3 (lower panel) ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in A (n=3). (C) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells. (D) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT N543 55Q with the indicated kinase for 24 hour in HEK293T cells from the experiment in C (representative of 3 repeats). (E) Representative western blot of HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells. (F) Quantification of HTT pS13 using the Singulex assay (using ab-MW1 as capture and CHDI-90001039 or 2B7 as detection antibodies for pS13 or total HTT) upon coexpression of HTT-FL 48Q or HTT-FL 48Q S13A/S16A with the indicated kinase for 24 hour in HEK293T cells from the experiments likein E (n=3). (G) Representative western blot of immunoprecipitated (IP) HTT (ab-MAB2166) and HTT pS13 (ab-CHDI-90001039-1) after lentivirus (LV)-mediated overexpression of TBK1 or TBK1 KD for 72 hour in rat primary striatal neuronal cells. (H) Quantification of the fold changes in the HTT pS13 ratio to total HTT compared to those of the kinase-dead mutant from the experiments like in G (n=3).


Figure 3 ( A ) Representative western blot of soluble HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation - incompetent mutants with the indicated kinases for 48 hour in HEK293T cells . ( B ) The graph indicates the fold change in HTT in samples like illustrated in A , relative to empty vector controls normalized to GAPDH . ( C ) Representative immunoblot of the HTT ( ab - MAB5492 ) after filter retardation assay from the insoluble cellular fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation - incompetent mutants with the indicated kinases for 48 hour in HEK293T cells . ( D ) Quantification of the fold change in HTT aggregates compared to EV expressing HTTex1 16Q from the experiments like in C . ( E ) Representative immunofluorescence images of HTT aggregates ( eGFP ) and TBK1 ( ab - anti - MYC ) upon coexpression of HTTex1 72Q eGFP or its phosphorylation - incompetent mutants with the indicated kinases for 48 hour in HEK293T cells ( scale bar 20μm ) . ( F ) Quantification of the percentage of co - transfected cells presenting aggregates upon expression of the indicated kinase , from the experiments like in E . ( G ) Quantification of the percentage of cells presenting nuclear aggregates from the experiments like in E . ( H ) Representative western blots of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) in soluble fraction and aggregates from HEK293T cells transfected first with HTTex1 72Q eGFP and then after 48 hour ( to enable the formation of aggregates before the kinase expression ) ; with TBK1 ; cells were lysed at the indicated time after kinase transfection . ( I ) Quantification of the percentage of cells presenting aggregates and kinase expression from the experiments in H .

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Figure 3(A) Representative western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (B) The graph indicates the fold change in HTT in samples like illustrated in A, relative to empty vector controls normalized to GAPDH. (C) Representative immunoblot of the HTT (ab-MAB5492) after filter retardation assay from the insoluble cellular fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (D) Quantification of the fold change in HTT aggregates compared to EV expressing HTTex1 16Q from the experiments like in C. (E) Representative immunofluorescence images of HTT aggregates (eGFP) and TBK1 (ab-anti-MYC) upon coexpression of HTTex1 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells (scale bar 20μm).(F) Quantification of the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in E. (G) Quantification of the percentage of cells presenting nuclear aggregates from the experiments like in E. (H) Representative western blots of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) in soluble fraction and aggregates from HEK293T cells transfected first with HTTex1 72Q eGFP and then after 48 hour (to enable the formation of aggregates before the kinase expression); with TBK1; cells were lysed at the indicated time after kinase transfection. (I) Quantification of the percentage of cells presenting aggregates and kinase expression from the experiments in H.


Figure 4 ( A Quantification of cell death by a nuclear condensation assay , cells were fixed and the nuclei were stained with Hoechst dye , cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead . ( A ) The percentage of cell death at DIV14 , after co - transfection of indicated kinases and HTTex1 plasmid in rat primary striatal neurons at DIV9 ( n = 3 ) , B ) Quantification of cell death by a nuclear condensation assay , cells were fixed and the nuclei were stained with Hoechst dye , cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead . ( B ) The percentage of cell death at DIV7 , after co - transfection of indicated kinases and HTT N586 plasmid in mouse primary striatal neurons at DIV5 ( n = 8 ) . ( C ) Quantification of cell death by a TUNEL assay in rat primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid . The cells were fixed at DIV14 , and the percentage of TUNEL - positive neurons was counted and plotted ( n = 3 ) . ( D ) The percentage of cell death . , quantification by a nuclear condensation assay in mouse primary cortical neurons co - transfected with the indicated HTT N586 and kinases at DIV5 , cells were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7 . Cells were fixed , and the nuclei were stained with Hoechst dye ( n = 3 ) . ( E ) Quantification of cell death by a TUNEL assay in mouse primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid . The cells were fixed on DIV14 , and the percentage of TUNEL - positive neurons was counted and plotted ( n = 3 ) .

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 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 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

Figure 4(A Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (A) The percentage of cell death at DIV14, after co-transfection of indicated kinases and HTTex1 plasmid in rat primary striatal neurons at DIV9 (n=3),B) Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (B) The percentage of cell death at DIV7, after co-transfection of indicated kinases and HTT N586 plasmid in mouse primary striatal neurons at DIV5 (n=8).(C) Quantification of cell death by a TUNEL assay in rat primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed at DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3).(D) The percentage of cell death., quantification by a nuclear condensation assay in mouse primary cortical neurons co-transfected with the indicated HTT N586 and kinases at DIV5, cells were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7. Cells were fixed, and the nuclei were stained with Hoechst dye (n=3).(E) Quantification of cell death by a TUNEL assay in mouse primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed on DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3).


Figure 5 ( A ) Representative immunofluorescence image of C . elegans overexpressing HTT N513 Q15 and HTT N513 Q128 ( scale bar 20μm ) . Top panel complete worm , lower panel head region . ( B ) Western blot showing HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) levels in C . elegans overexpressing HTT N513 Q15 and HTT N513 Q128 . ( C ) Quantification of the fold changes in ratios of HTT pS13 and pT3 to total HTT in C . elegans overexpressing HTT N513 Q15 and HTT N513 Q128 from the experiments like in B ( n = 3 ) . ( D ) Representative western blots showing the HTT ( ab - MAB5492 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) levels from the detergent soluble fraction and HTT aggregates ( ab - MAB5492 ) from the detergent insoluble fraction in three different transgenic C . elegans lines overexpressing TBK1 or the TBK1 KD . ( E ) The bottom graph indicates the fold change in the HTT pS13 ( ab - CHDI - 90001039 - 1 ) ratios to total HTT ( ab - MAB5492 ) compared to those in the kinase - dead mutant line 1 from the experiments like in D . The top graph indicates the fold change in HTT aggregates ( ab - MAB5492 ) compared to that in the kinase - dead mutant the experiments like in D ( n = 3 ) . ( F ) Mobility of the Q15 and Q128 C . elegans lines upon overexpression of TBK1 or TBK1 KD ( n = 26 - 29 ) . ( G ) Survival graph of the Q15 and Q128 C . elegans lines upon overexpression of TBK1 or TBK1 KD ( n = 30 ) . ( H ) Representative western blots showing HTT ( ab - MAB5492 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) from the detergent soluble fraction and HTT aggregates ( ab - MAB5492 ) from the detergent insoluble fraction in TBK1 orthologue ( ikke - 1 or unc - 51 ) RNAi - treated C . elegans lines expressing HTT N513 Q15 and HTT N513 Q128 . ( I ) Quantification of the fold change in the ratio of pS13 HTT to total HTT compared to non - targeting RNAi from the experiments like in H ( n = 4 ) .

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 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 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 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

Figure 5(A) Representative immunofluorescence image of C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 (scale bar 20μm). Top panel complete worm, lower panel head region.(B) Western blot showing HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) levels in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128. (C) Quantification of the fold changes in ratios of HTT pS13 and pT3 to total HTT in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 from the experiments like in B (n=3). (D) Representative western blots showing the HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) levels from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in three different transgenic C. elegans lines overexpressing TBK1 or the TBK1 KD. (E) The bottom graph indicates the fold change in the HTT pS13 (ab-CHDI-90001039-1) ratios to total HTT (ab-MAB5492) compared to those in the kinase-dead mutant line 1 from the experiments like in D. The top graph indicates the fold change in HTT aggregates (ab-MAB5492) compared to that in the kinase-dead mutant the experiments like in D (n=3). (F) Mobility of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n= 26-29).(G) Survival graph of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n=30).(H) Representative western blots showing HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in TBK1 orthologue (ikke-1 or unc-51) RNAi-treated C. elegans lines expressing HTT N513 Q15 and HTT N513 Q128. (I) Quantification of the fold change in the ratio of pS13 HTT to total HTT compared to non-targeting RNAi from the experiments like in H (n=4).


Figure 6 ( A ) Representative immunoblot of a filter retardation of HTT ( ab - MAB5492 ) from the insoluble cellular fraction ; HEK293T cells co - expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour , and for the last 16 hour , they were treated with the indicated proteasome inhibitor ( MG132 , 5 µM ) or autophagy inhibitor ( Baf A1 , 200 nM , NH4Cl , 10 mM , 3 - MA , 5 mM ) . ( B ) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A ( n = 3 ) . ( C ) Immunoblot of LC3 ( ab48394 ) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour ; for the last hour , Baf A1 ( 500 nM ) was added as indicated . ( D ) Fold change in LC3 - II levels ( lower band ) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C ( n = 3 ) . ( E ) Immunoblot of LC3 ( ab48394 ) from soluble rat primary neuronal cells overexpressing lentivirus - mediated TBK1 and TBK1 KD for 96 hour . For the last 1 or 4 hour , Baf A1 ( 500 nM ) was added as indicated . ( F ) Quantification of the fold change in LC3 - II levels ( lower band ) compared to the kinase dead mutant , which was untreated , normalized to GAPDH from the blots like in E ( n = 3 ) .

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 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

Figure 6(A) Representative immunoblot of a filter retardation of HTT (ab-MAB5492) from the insoluble cellular fraction; HEK293T cells co-expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour, and for the last 16 hour, they were treated with the indicated proteasome inhibitor (MG132, 5 µM) or autophagy inhibitor (Baf A1, 200 nM, NH4Cl, 10 mM, 3-MA,5 mM). (B) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A (n=3).(C) Immunoblot of LC3 (ab48394) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour; for the last hour, Baf A1 (500 nM) was added as indicated. (D) Fold change in LC3-II levels (lower band) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C (n=3). (E) Immunoblot of LC3 (ab48394) from soluble rat primary neuronal cells overexpressing lentivirus-mediated TBK1 and TBK1 KD for 96 hour. For the last 1 or 4 hour, Baf A1 (500 nM) was added as indicated. (F) Quantification of the fold change in LC3-II levels (lower band) compared to the kinase dead mutant, which was untreated, normalized to GAPDH from the blots like in E (n=3).


Figure 7 ( A ) Western blot of soluble HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT aggregates ( ab - MAB5492 ) , upon coexpression of the HTTex1 72Q eGFP or HTTex1 72Q eGFP S13A / S16A variant with TBK1 KD , TBK1 or TBK1 Δ690 - 713 for 48 hour in HEK293 cells . ( B ) The graph indicates the calculated fold changes in HTT , HTT pS13 ratio to total HTT and HTT aggregates all compared to the levels in TBK1 KD from the experiments like in A ( bottom panel normalized to Tubulin , n = 3 ) . ( C ) Representative immunofluorescence images of eGFP HTTex1 72Q or its phosphorylation - deficient variant S13A / S16A upon coexpression with TBK1 KD or TBK1 or TBK1 Δ690 - 713 ( ab - anti - Myc ) with for 48 hour in HEK293 cells ( scale bar 20μm ) . ( D ) The graph indicates the percentage of co - transfected cells presenting aggregates upon expression of the indicated kinase , from the experiments like in C ( n = 3 ) .

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

Figure 7(A) Western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT aggregates (ab-MAB5492), upon coexpression of the HTTex1 72Q eGFP or HTTex1 72Q eGFP S13A/S16A variant with TBK1 KD, TBK1 or TBK1 Δ690-713 for 48 hour in HEK293 cells. (B) The graph indicates the calculated fold changes in HTT, HTT pS13 ratio to total HTT and HTT aggregates all compared to the levels in TBK1 KD from the experiments like in A (bottom panel normalized to Tubulin, n=3). (C) Representative immunofluorescence images of eGFP HTTex1 72Q or its phosphorylation-deficient variant S13A/S16A upon coexpression with TBK1 KD or TBK1 or TBK1 Δ690-713 (ab-anti-Myc) with for 48 hour in HEK293 cells (scale bar 20μm). (D) The graph indicates the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in C (n=3).


Figure 1A - O ( A , F , K ) slices through electron cryo - tomograms of L . pneumophila , P . aeruginosa , and S . oneidensis cells , respectively , highlighting a PL sub - complex in the outer membrane ( dashed yellow circle ) . ( B , G , L ) Enlarged views of the complexes . ( C , H , M ) Sub - tomogram averages of PL sub - complexes from each species . ( D , I , N ) Schematic representations of the sub - tomogram averages with different rings colored and labeled . ( E , J , O ) Sub - tomogram averages of fully - assembled flagella from each species for comparison . Scale bars : ( black ) 50 nm , ( orange ) 25 nm , ( red ) 20 nm .

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

Figure 1A-O (A, F, K) slices through electron cryo-tomograms of L. pneumophila, P. aeruginosa, and S. oneidensis cells, respectively, highlighting a PL sub-complex in the outer membrane (dashed yellow circle). (B, G, L) Enlarged views of the complexes. (C, H, M) Sub-tomogram averages of PL sub-complexes from each species. (D, I, N) Schematic representations of the sub-tomogram averages with different rings colored and labeled. (E, J, O) Sub-tomogram averages of fully-assembled flagella from each species for comparison. Scale bars: (black) 50 nm, (orange) 25 nm, (red) 20 nm.