Figure 5U2OS cells were transfected with a Flag - DDX5 expression vector in the presence of siCTL or siPRMT5 . Lysates were immunoprecipitated with IgG or anti - Flag antibodies as indicated . The WCE and the bound proteins were Western blotted with anti - symmetrical dimethylarginine antibody or anti - Flag antibodies . Coomassie blue ( left panel ) and in vitro methylation assay ( right panel ) of indicated GST - DDX5 fragments and the GST - DDX5 - RK mutant . Immunofluorescence analysis with S9 . 6 and anti - Flag antibodies of U2OS cells transfected with Flag - tagged DDX5 , DDX5 - RK or DDX5 - XD ( helicase dead ) . Nuclear S9 . 6 signal was counted in both Flag - negative and Flag - positive cells . The Flag - negative cells were considered as untransfected cells . The graphs shown represent the quantification with the S . E . M from 3 independent experiments . Statistical significance was assessed using one - way ANOVA t - test . * * * * , p < 0 . 0001 . Immunofluorescence analysis with S9 . 6 and anti - Flag antibodies of U2OS cells transfected with siCTL or siDDX5 - 1 and Flag - tagged DDX5 ( + WT ) or DDX5 - RK ( + RK ) as indicated . The graphs show the average and S . E . M . from three independent experiments . Statistical significance was assessed using one - way ANOVA t - test . * * * * , p < 0 . 0001 . HEK293 cells were transfected with siCTL , or siDDX5 . The next day , the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector ( - ) , expression vectors encoding Flag - DDX5 ( WT ) , or Flag - DDX5 - RK ( RK ) . Forty eight hours later , all the cells were subjected to DRIP - qPCR analysis . The average with the S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and n . s . : not significant .

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

Figure 5U2OS cells were transfected with a Flag-DDX5 expression vector in the presence of siCTL or siPRMT5. Lysates were immunoprecipitated with IgG or anti-Flag antibodies as indicated. The WCE and the bound proteins were Western blotted with anti-symmetrical dimethylarginine antibody or anti-Flag antibodies.Coomassie blue (left panel) and in vitro methylation assay (right panel) of indicated GST-DDX5 fragments and the GST-DDX5-RK mutant.Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with Flag-tagged DDX5, DDX5-RK or DDX5-XD (helicase dead). Nuclear S9.6 signal was counted in both Flag-negative and Flag-positive cells. The Flag-negative cells were considered as untransfected cells. The graphs shown represent the quantification with the S.E.M from 3 independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.Immunofluorescence analysis with S9.6 and anti-Flag antibodies of U2OS cells transfected with siCTL or siDDX5-1 and Flag-tagged DDX5 (+WT) or DDX5-RK (+RK) as indicated. The graphs show the average and S.E.M. from three independent experiments. Statistical significance was assessed using one-way ANOVA t-test. ****, p < 0.0001.HEK293 cells were transfected with siCTL, or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). Forty eight hours later, all the cells were subjected to DRIP-qPCR analysis. The average with the S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and n.s.: not significant.


Figure 6HEK293 cells were transfected with empty pcDNA3 vector ( - ) or Flag - DDX5 ( + ) . Whole cell lysates ( WCE ) and anti - Flag immunoprecipitations ( IP ) were immunoblotted with anti - Flag and anti - XRN2 antibodies . Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G ( IgG ) or anti - XRN2 antibodies . The bound proteins were separated by SDS - PAGE followed by Western blotting with anti - DDX5 or anti - XRN2 antibodies . The migration of DDX5 and XRN2 is depicted with an arrow . U2OS cells were transfected with empty pcDNA3 vector ( pcDNA3 ) or expression vectors encoding Flag - DDX5 WT ( 1 - 614 ) or truncated Flag - DDX5 proteins ( 60 - 614 ; 1 - 554 ; 1 - 435 ) . WCE of the transfected cells ( left panel ) were Western blotted with anti - Flag and anti - XRN2 antibodies to confirm equivalent expression . The transfected cells were lysed and anti - Flag immunoprecipitations ( right panel ) performed for the presence of co - immunoprecipitating endogenous XRN2 by Western blotting with anti - XRN2 antibodies . M denotes molecular mass markers in kDa . Untransfected and U2OS stably expressing Flag - DDX5 WT or Flag - DDX5 - RK were subjected to immunoprecipitation with anti - Flag antibody . The WCE and the anti - Flag immunoprecipitated proteins were Western blotted with anti - monomethylarginine ( MeR ) , anti - Flag and anti - XRN2 antibodies , respectively . M denotes molecular mass markers in kDa . U2OS cells transfected with siCTL , siDDX5 , or siXRN2 were subjected to DRIP - qPCR analysis with anti - IgG , and anti - S9 . 6 antibodies with or without RNaseH treatment . The average and S . E . M . from 3 independent experiments is shown . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 .

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

Figure 6HEK293 cells were transfected with empty pcDNA3 vector (-) or Flag-DDX5 (+). Whole cell lysates (WCE) and anti-Flag immunoprecipitations (IP) were immunoblotted with anti-Flag and anti-XRN2 antibodies.Untransfected U2OS cells were lyzed and subjected to immunoprecipitation with control immunoglobulin G (IgG) or anti-XRN2 antibodies. The bound proteins were separated by SDS-PAGE followed by Western blotting with anti-DDX5 or anti-XRN2 antibodies. The migration of DDX5 and XRN2 is depicted with an arrow.U2OS cells were transfected with empty pcDNA3 vector (pcDNA3) or expression vectors encoding Flag-DDX5 WT (1-614) or truncated Flag-DDX5 proteins (60-614; 1-554; 1-435). WCE of the transfected cells (left panel) were Western blotted with anti-Flag and anti-XRN2 antibodies to confirm equivalent expression. The transfected cells were lysed and anti-Flag immunoprecipitations (right panel) performed for the presence of co-immunoprecipitating endogenous XRN2 by Western blotting with anti-XRN2 antibodies. M denotes molecular mass markers in kDa.Untransfected and U2OS stably expressing Flag-DDX5 WT or Flag-DDX5-RK were subjected to immunoprecipitation with anti-Flag antibody. The WCE and the anti-Flag immunoprecipitated proteins were Western blotted with anti-monomethylarginine (MeR), anti-Flag and anti-XRN2 antibodies, respectively. M denotes molecular mass markers in kDa.U2OS cells transfected with siCTL, siDDX5, or siXRN2 were subjected to DRIP-qPCR analysis with anti-IgG, and anti-S9.6 antibodies with or without RNaseH treatment. The average and S.E.M. from 3 independent experiments is shown. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.


Figure 7HEK293 cells were transfected with siCTL , siDDX5 , or siXRN2 . The gene location and qPCR amplification region are shown at the top of each panel . The identified R - loop peaks were extracted from R - loop database ( R - loop DB ) for each gene region . R - loop : DRIP denotes anti - S9 . 6 DRIP - qPCR and Pol II ChIP denotes RNA pol II ChIP . The bar graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 . B , E , H , S and X denote the location of the BsrGI , EcoRI , HindIII , SspI , and XbaI .

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

Figure 7HEK293 cells were transfected with siCTL, siDDX5, or siXRN2. The gene location and qPCR amplification region are shown at the top of each panel. The identified R-loop peaks were extracted from R-loop database (R-loop DB) for each gene region. R-loop: DRIP denotes anti-S9.6 DRIP-qPCR and Pol II ChIP denotes RNA pol II ChIP. The bar graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001. B, E, H, S and X denote the location of the BsrGI, EcoRI, HindIII, SspI, and XbaI.


Figure 8Schematic representation of the β - actin locus and the location of the genomic PCR primers used for ChIP - qPCR and DRIP - qPCR . Prom ( promoter ) , RB ( region B ) , poly ( A ) and RD ( region D ) indicate the regions analyzed by qPCR . HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed . The XRN2 ChIP signal was normalized to the Pol II ChIP signal . WCE were immunoblotted with anti - DDX5 and α - tubulin antibodies , the latter was used as a loading control . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 . HEK293 cells were transfected with siCTL , siDDX5 or siXRN2 . The cells were subjected to Western blotting , RNA Pol II ChIP and DRIP analysis of β - actin gene . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 . HEK293 cells were transfected with siCTL or siDDX5 . The next day , the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector ( - ) , expression vectors encoding Flag - DDX5 ( WT ) , or Flag - DDX5 - RK ( RK ) . The cells were subjected to Western blotting to visualize Flag - DDX WT and RK expression . Pol II ChIP and DRIP analysis of β - actin gene were performed . For the ChIP analysis , the Y - axis shows the signal - to - noise ratio of RNA Pol II IP relative to control IgG IP . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 . HEK293 cells were transfected with siCTL or siPRMT5 . The cells were subjected to Western blotting , RNA Pol II ChIP and DRIP analysis of β - actin gene . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 . HEK293 cells were transfected with empty plasmid ( - ) , wild type Flag - DDX5 ( WT ) or its RK mutant ( RK ) and subjected to western blot and Flag - DDX5 ChIP - qPCR analysis of β - actin gene . The graphs show the average and S . E . M . from at least three independent experiments . Statistical significance was assessed using Student ' s t - test . * : p < 0 . 05 ; * * : p < 0 . 01 and * * * * : p < 0 . 0001 ; n . s . not significant .

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

Figure 8Schematic representation of the β-actin locus and the location of the genomic PCR primers used for ChIP-qPCR and DRIP-qPCR. Prom (promoter), RB (region B), poly(A) and RD (region D) indicate the regions analyzed by qPCR.HEK293 cells were transfected with siCTL or siDDX5 and XRN2 ChIP assays were performed. The XRN2 ChIP signal was normalized to the Pol II ChIP signal. WCE were immunoblotted with anti-DDX5 and α-tubulin antibodies, the latter was used as a loading control. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.HEK293 cells were transfected with siCTL, siDDX5 or siXRN2. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.HEK293 cells were transfected with siCTL or siDDX5. The next day, the cells that received siDDX5 were subsequently transfected with empty pcDNA3 vector (-), expression vectors encoding Flag-DDX5 (WT), or Flag-DDX5-RK (RK). The cells were subjected to Western blotting to visualize Flag-DDX WT and RK expression. Pol II ChIP and DRIP analysis of β-actin gene were performed. For the ChIP analysis, the Y-axis shows the signal-to-noise ratio of RNA Pol II IP relative to control IgG IP. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001.HEK293 cells were transfected with siCTL or siPRMT5. The cells were subjected to Western blotting, RNA Pol II ChIP and DRIP analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01.HEK293 cells were transfected with empty plasmid (-), wild type Flag-DDX5 (WT) or its RK mutant (RK) and subjected to western blot and Flag-DDX5 ChIP-qPCR analysis of β-actin gene. The graphs show the average and S.E.M. from at least three independent experiments. Statistical significance was assessed using Student's t-test. *: p < 0.05; **: p < 0.01 and ****: p < 0.0001; n.s. not significant.


Figure 1In vitro niche dependency of HGSOC tumor cells . Phase - contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth , while they do not grow at all in Wnt3a supplemented medium . Also , inhibition of BMP signaling through Noggin has strong negative effect on the initial growth . Scale bar : 500 µm . E - EGF , F - FGF10 , N - Noggin , R - R - spondin1 , B - Basic medium , P - PassageCancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines . Scale bar : 20 µm . HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization . Scale bar : 100 µm . HGSOC organoids show differential response to carboplatin treatment , confirming patient - specific sensitivity of the cultures . Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors . Data represent mean ± SD of technical triplicates .

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

Figure 1In vitro niche dependency of HGSOC tumor cells. Phase-contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 µm. E - EGF, F - FGF10, N -Noggin, R - R-spondin1, B - Basic medium, P - PassageCancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 µm.HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization. Scale bar: 100 µm.HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.


Figure 2Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long - term organoid culture . Color code indicates type of mutations which were detected . Representative western blot showing loss or overexpression of p53 in individual organoid lines . Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain - of - function TP53 mutation . Scale bars : 100 µm ( Tissue ) and 50 µm ( Organoid ) . Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue . Data represent the median , quartiles , maximum , and minimum of the normalized expression from 8 different donors . Heat map of differentially expressed genes between cancer and healthy tissue / organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples . Differential expression determined by single - color microarray for 8 different patient samples was significant for all genes with p < 0 . 05 except for OVGP1 and TOP2A in organoids .

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Figure 2Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long-term organoid culture. Color code indicates type of mutations which were detected.Representative western blot showing loss or overexpression of p53 in individual organoid lines.Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid).Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue. Data represent the median, quartiles, maximum, and minimum of the normalized expression from 8 different donors.Heat map of differentially expressed genes between cancer and healthy tissue/organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples. Differential expression determined by single-color microarray for 8 different patient samples was significant for all genes with p < 0.05 except for OVGP1 and TOP2A in organoids.


Figure 3Experimental approach for genetic manipulation of FT epithelial cells ( FTECs ) . FTECs were sequentially transduced in 2D culture with replication - deficient viruses containing specific shRNAs and different selection markers . After sorting cells were seeded in Matrigel for organoid formation . Scale bars : 2 cm ( tissue ) , 200 µm ( 2D culture ) and 500 µm ( 3D culture ) . Confirmation of robust knockdowns of p53 , PTEN and RB in FT organoids on RNA and protein level . Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ± SEM from 3 different donors . A representative western blot from one donor is depicted . RNA as well as protein samples were taken at passage 1 or 2 of each organoid line . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild typeCyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis . Confocal images of triple KD organoids reveal increased DNA damage ( yH2AX , marked by asterisks ) , atypic nuclei ( DRAQ5 ) and loss of apicobasal polarity . Scale bar : 20µm . Confirmation of robust knockdowns of p53 , PTEN and RB in FT organoids on RNA and protein level . Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ± SEM from 3 different donors . A representative western blot from one donor is depicted . RNA as well as protein samples were taken at passage 1 or 2 of each organoid line . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis . Presence of ciliated cells ( arrowheads ) , as revealed by immunostaining against detyrosinated Tubulin ( dtyrTub ) in triple KD organoids , proves capacity for terminal differentiation . Scale bar : 50µm .

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 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 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

Figure 3Experimental approach for genetic manipulation of FT epithelial cells (FTECs). FTECs were sequentially transduced in 2D culture with replication-deficient viruses containing specific shRNAs and different selection markers. After sorting cells were seeded in Matrigel for organoid formation. Scale bars: 2 cm (tissue), 200 µm (2D culture) and 500 µm (3D culture). Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild typeCyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis.Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm.Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis. Presence of ciliated cells (arrowheads), as revealed by immunostaining against detyrosinated Tubulin (dtyrTub) in triple KD organoids, proves capacity for terminal differentiation. Scale bar: 50µm.


Figure 4In long - term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234 . Scale bar : 500 µm . Growth curves and phase - contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long - term growth capacity . Scale bar : 500 µm . Levels of DNA damage ( yH2AX ) and apoptosis ( cleaved caspase - 3 , PARP1 ) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls . Triple KD ( p53 / PTEN / RB ) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p < 0 . 00005 . Differential expression of stemness - and differentiation - related genes of triple KD organoids grown in OCM vs . FTM . Fold - changes were derived from microarray data and indicate and an increase in stemness ( CD133 , SOX2 , MYCN ) and a drop in differentiation ( OVGP1 , PGR , FOXJ1 ) under OCM conditions . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p < 0 . 05 . FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM , suggesting a decrease in differentiation capacity . Data represent the mean ± SEM from technical triplicates for two independent knockdown cultures . Proportion of ciliated cells ( detyrosinated Tubulin ; dtyrTub ) is significantly reduced in KD organoids under OCM growth conditions , confirming inhibition of differentiation as illustrated by confocal images ( Scale bar : 50 µm ) . The quantification plot depicts the mean ± SEM from 3 independent biological replicates , based on quantification of the average number of ciliated cells per counted nuclei . 1000 cells were counted per individual experiment . * p < 0 . 05 , two - sided Student ' s t - test .

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Figure 4In long-term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234. Scale bar: 500 µm.Growth curves and phase-contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long-term growth capacity. Scale bar: 500 µm.Levels of DNA damage (yH2AX) and apoptosis (cleaved caspase-3, PARP1) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls.Triple KD (p53/PTEN/RB) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.00005.Differential expression of stemness- and differentiation-related genes of triple KD organoids grown in OCM vs. FTM. Fold-changes were derived from microarray data and indicate and an increase in stemness (CD133, SOX2, MYCN) and a drop in differentiation (OVGP1, PGR, FOXJ1) under OCM conditions. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.05.FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM, suggesting a decrease in differentiation capacity. Data represent the mean ±SEM from technical triplicates for two independent knockdown cultures.Proportion of ciliated cells (detyrosinated Tubulin; dtyrTub) is significantly reduced in KD organoids under OCM growth conditions, confirming inhibition of differentiation as illustrated by confocal images (Scale bar: 50 µm). The quantification plot depicts the mean ±SEM from 3 independent biological replicates, based on quantification of the average number of ciliated cells per counted nuclei. 1000 cells were counted per individual experiment. *p < 0.05, two-sided Student's t-test.


Figure 5The proportion of CD133 + cells was determined by FACS analysis after growing triple KD and control cells in FTM vs . OCM for two passages . The graph is representative of two biological replicates . Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number ( representative graph of 3 independent experiments ) . The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ± SEM . * p < 0 . 05 , two - sided Student ` s t - test ; KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild typePhase - contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists ( Wnt3a / RSPO1 ) over 2 passages ( Scale bar : 500µm ) . Differences in cell number were confirmed by the respective cell viability assays ( performed in technical triplicates ) . Data represent the mean ± SEM for 5 different OC organoid lines ( n = 5 ) . * * * p < 0 . 001 , two - sided Student ` s t - test . The number of CD133 + cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a / RSPO1 and with or without Noggin over 2 passages , confirms a sharp drop in stemness in the presence of Wnt agonists . Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids , proving that the growth inhibitory effect is due to the presence of Wnt agonists . The bar plot represents the mean ± SEM of triplicates performed with a luminescent cell viability assay . * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ` s t - test .

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Figure 5The proportion of CD133+ cells was determined by FACS analysis after growing triple KD and control cells in FTM vs. OCM for two passages. The graph is representative of two biological replicates.Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number (representative graph of 3 independent experiments). The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ±SEM. * p < 0.05, two-sided Student`s t-test; KD - Knockdown, Vec Ctrl - Vector Control, WT - Wild typePhase-contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists (Wnt3a/RSPO1) over 2 passages (Scale bar: 500µm). Differences in cell number were confirmed by the respective cell viability assays (performed in technical triplicates). Data represent the mean ±SEM for 5 different OC organoid lines (n=5). *** p < 0.001, two-sided Student`s t-test.The number of CD133+ cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a/RSPO1 and with or without Noggin over 2 passages, confirms a sharp drop in stemness in the presence of Wnt agonists.Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids, proving that the growth inhibitory effect is due to the presence of Wnt agonists. The bar plot represents the mean ±SEM of triplicates performed with a luminescent cell viability assay. * p < 0.05, *** p < 0.001, two-sided Student`s t-test.


Figure 6Comparative analysis of microarray data revealed consistent up - regulation of MYCN , the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1 , AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids . Differential expression was determined either by single - color microarray for the cancer samples ( 8 replicates ) or dual - color microarray for the knockdowns ( 2 replicates ) and is significant for all genes with p < 0 . 05 . Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs . FTM conditions . Depicted is the mean ± SEM of the normalized MYCN mRNA expression level of three biological replicates ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 , two - sided Student ` s t - test . Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p ≤ 0 . 005 . FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples . Data represent the mean ± SEM of technical triplicates .

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 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 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

Figure 6Comparative analysis of microarray data revealed consistent up-regulation of MYCN, the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1, AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids. Differential expression was determined either by single-color microarray for the cancer samples (8 replicates) or dual-color microarray for the knockdowns (2 replicates) and is significant for all genes with p < 0.05.Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs. FTM conditions. Depicted is the mean ±SEM of the normalized MYCN mRNA expression level of three biological replicates (n=3). ** p < 0.01, *** p < 0.001, two-sided Student`s t-test.Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p ≤ 0.005.FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.


Figure 1A ) Perturbation in the free energy of PKD2L1 opening ( ∆ G ° ) and difference in closure rates ( ∆ τclose ) inducted by substitutions within the pore module . Error bars = S . D as estimated by the propagation of error equation , as descried in statistics subsection .

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Figure 1A) Perturbation in the free energy of PKD2L1 opening (∆G°) and difference in closure rates (∆τclose) inducted by substitutions within the pore module. Error bars = S.D as estimated by the propagation of error equation, as descried in statistics subsection.


Figure 2B ) Exemplar tail currents from mutant channels where the energetics of opening are disassociated with the rate of channel closure - mutations which cause slower closure ( + ∆ τclose ) while also increasing the free energy opening ( + ∆ G ° ) .

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Figure 2B) Exemplar tail currents from mutant channels where the energetics of opening are disassociated with the rate of channel closure- mutations which cause slower closure (+∆τclose) while also increasing the free energy opening (+∆G°).


Figure 3D ) Comparing voltage - dependent opening based on normalized tail currents measured from WT , single and double mutant channels . Top , single and double mutant channels including mutations to the outer and inner gate . Middle and bottom , control groups comparing tail currents from non - gate S4 - S5 linker mutations ( D476A and G479A ) and those from the outer and inner gates . Error bars indicate standard deviation for as estimated by the propagation of error equation , as descried in statistics subsection . Number cells tested ( N ) are indicated in parentheses for each group . E ) The coupling energy ( ∆ ∆ G ° ) resulting from the mutant cycle analysis resulting from the data shown in D . Error bars indicate standard deviation for as estimated by the propagation of error equation , as descried in statistics subsection . Number cells tested ( N ) are indicated in parentheses for each group .

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Figure 3D) Comparing voltage-dependent opening based on normalized tail currents measured from WT, single and double mutant channels. Top, single and double mutant channels including mutations to the outer and inner gate. Middle and bottom, control groups comparing tail currents from non-gate S4-S5 linker mutations (D476A and G479A) and those from the outer and inner gates. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.E) The coupling energy (∆∆G°) resulting from the mutant cycle analysis resulting from the data shown in D. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.


Figure 4B ) Summary of the change in voltage - dependent inactivation ( ∆ V1 / 2 Inact . ) of pore mutant channels . ∆ V1 / 2 Inact . , are plotted relative to the pore module topology of the channel . C ) Voltage - dependent inactivation of test pulse tail currents recorded from WT , single and double mutant channels .

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Figure 4B) Summary of the change in voltage-dependent inactivation (∆V1/2 Inact.) of pore mutant channels. ∆V1/2 Inact., are plotted relative to the pore module topology of the channel.C) Voltage-dependent inactivation of test pulse tail currents recorded from WT, single and double mutant channels.


Figure 5C ) Exemplar whole cell currents ( Top ) at the zero , three and seven minute time points . Current records ( Bottom ) from cells expressing the F514C : P538C and treated with extracellular 100 μM 1 , 2 - methanediyl bismethanethiosulfonate ( M2M ) . D ) Box ( S . E . M . ) and whisker ( S . D . ) plots comparing tail currents normalized to their initial amplitude at 3 minute ( Left ) and 7 - minute time points ( Right ) . N = 6 cells per group . Results from each individual cells are indicated by grey open circles and the average for the channel is indicated by a horizontal line . P - values results from a one - way ANOVA statistical analysis is shown above the plots . Dashed lines the level normalcy , no change in Itail relative to time point zero .

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Figure 5C) Exemplar whole cell currents (Top) at the zero, three and seven minute time points. Current records (Bottom) from cells expressing the F514C:P538C and treated with extracellular 100 μM 1,2-methanediyl bismethanethiosulfonate (M2M).D) Box (S.E.M.) and whisker (S.D.) plots comparing tail currents normalized to their initial amplitude at 3 minute (Left) and 7-minute time points (Right). N = 6 cells per group. Results from each individual cells are indicated by grey open circles and the average for the channel is indicated by a horizontal line. P-values results from a one-way ANOVA statistical analysis is shown above the plots. Dashed lines the level normalcy, no change in Itail relative to time point zero.


Figure 1Left : Fluorescence changes of IRDye700 labelled dsDNA ( at 125 nM ) when incubated at increasing ID2 ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FANCD2 ( D2Ub ) concentrations ( ranging from 1 . 3 nM to 5 . 9 µM ) . Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from model fitting . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 23 - 24 data points each ) . Assessment of protein - DNA interactions using Electro - mobility shift assays ( EMSA ) . IRDye700 labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of non / single / double - ubiquitinated ID2 ( His6 - TEV - V5 - FANCI and FLAG - FANCD2 ) protein complexes ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FLAG - FANCD2 ( D2Ub ) . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . EMSA gels of ID2 complexes are representative of 3 replicate experiments .

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Figure 1Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each).Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.


Figure 2Left : CryoEM 2D classes of ID2Ub and IUbD2Ub - dsDNA . Right : 3D reconstructions of ID2Ub ( EMD - 10843 ) and IUbD2Ub - DNA ( EMD - 10844 ) . Both structures exhibit a torus - like shape .

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Figure 2Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape.


Figure 3Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI can be efficiently ubiquitinated in the presence of DNA . Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA . Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI efficiently associate with FANCD2 . Fluorescence changes occurring when RED - tris - NTA - labelled FANCI ( IWT or IR1285Q ; both at 60 nM ) is incubated at increasing concentrations of FANCD2 ( ranging from 2 . 48 nM to 5 . 08 µM ) . Titrations were conducted twice ( two technical replicates ) and all data points for each protein - combination were used in fitting of a one - site binding model . Apparent Kd values ( mean ± SEM ) derived from fitting of a one - site binding model to the 24 data points of the two technical replicates are shown . FANCD2 within an IR1285QD2 complex is resistant to ubiquitination . Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA . Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS - PAGE . FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by PIFE . Left : Fluorescence changes when IRD700 - labelled dsDNA ( at 125 nM ) is incubated at increasing concentrations of IR1285Q + D2 or IR1285Q + D2Ub ( complex concentrations ranging from 1 . 54 nM to 3 . 8 µM ) . Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from the one - site binding model . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 22 data points each ) . IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values ( from data points shown in Figure 1A ) are also shown for comparison . FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by EMSAs . IRDye700 - labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of ID2 ( His6 - FANCI or His6 - FANCIR1285Q mixed with non - ubiquitinated or ubiquitinated FLAG - FANCD2 . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . Gels shown are representative of two replicate experiments .

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Figure 3Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA.Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE.FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison.FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.


Figure 4USP1 - UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI ( I ) , but not in the presence of ubiquitinated FANCI ( IUb ) . Ubiquitinated FANCD2 ( D2Ub ) was mixed with a 50 base pair dsDNA and either His6 - TEV - V5 - FANCI ( I ) , ubiquitinated His6 - TEV - V5 - FANCI ( IUb ) or no protein ; protein - DNA mixes were subsequently incubated with USP1 - UAF1 ( at 25 nM or 100 nM ) for indicated time periods . Deubiquitination of D2Ub and IUb was assessed , following SDS - PAGE , by Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody . USP7 and USP2 can deubiquitinate D2Ub , but their activity towards D2Ub is greatly reduced in the presence of FANCI ( I ) or ubiquitinated FANCI ( IUb ) . Reactions were set - up as in A , but with 100 nM USP7 or USP2 . Deubiquitination was assessed , following SDS - PAGE , by Coomassie staining . Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity / resistance to USP1 - UAF1 mediated deubiquitination of resulting products . Deubiquitination reactions in the presence or absence of USP1 - UAF1 ( 100 nM ) incubated for 30 minutes . Time course of USP1 - UAF1 mediated deubiquitination of IUbD2Ub - DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild - type ( WT ) ubiquitin . Progress of deubiquitination reaction was assessed following SDS - PAGE , by both Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody . FANCI ubiquitination with indicated ubiquitin - mutants ( or WT ) and subsequent deubiquitination of resulting IUbD2Ub - DNA complexes were conducted twice ; the residual FANCD2 ubiquitination , calculated from the FANCD2 blots for each time - point , was plotted for each type of ubiquitin in the protein complex , and corresponding deubiquitination progress curves were fitted using a one - phase decay model .

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Figure 4USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody.USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining.Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes.Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.


Figure 1 ( B ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg pQCXIP encoding IFITM3 variants containing N - terminal FLAG ( pQCXIP - FLAG - IFITM3 ) and immunostained with anti - FLAG M2 antibody . Scale bar , 5 μm . ( C ) Flow cytometry analysis of transfected cells in ( B ) immunostained with anti - FLAG M2 antibody . Corresponding mean flourescence intensity values are shown . ( D ) SDS - PAGE of cell lysates derived from 293T cells transfected in ( B ) followed by immunoblotting with anti - FLAG M2 and anti - actin antibodies . ( E ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T cells transfected with pQCXIP - FLAG - IFITM3 variants , HIV - 1 pNL4 - 3 , and Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 and anti - p24 Gag ( 183 - H12 - 5C ) . ( F ) Opti - prep purified supernatants from ( E ) were incubated with SupT1 target cells for 4 hours and virus - cell fusion was scored by flow cytometry . Mean + SD of 5 - 10 experiments ( each performed with virus produced from an independent transfection ) is shown . ( G ) Virus used in ( E ) were used to infect SupT1 cells and productive infection was scored at 48 - 60 hours by immunostaining with the KC57 antibody and flow cytometry . Expression of pCMV - MxA served as a negative control . The mean + SD of 5 - 10 experiments is shown . ( H ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg pQCXIP - FLAG - IFITM3 variants and immunostained with anti - FLAG M2 antibody ( I ) SDS - PAGE of cell lysates derived from 293T cells transfected in ( H ) followed by immunoblotting with anti - FLAG M2 and anti - actin antibodies . ( J ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T cells transfected with pQCXIP - FLAG - IFITM3 constructs , HIV - 1 pNL4 - 3 , and Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 and anti - p24 Gag . Scale bar , 5 μm . ( K ) Opti - prep purified supernatants from ( J ) were incubated with SupT1 target cells for 4 hours and virus - cell fusion was scored by flow cytometry . The mean + SD of 3 experiments is shown .

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Figure 1(B) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N-terminal FLAG (pQCXIP-FLAG-IFITM3) and immunostained with anti-FLAG M2 antibody. Scale bar, 5 μm.(C) Flow cytometry analysis of transfected cells in (B) immunostained with anti-FLAG M2 antibody. Corresponding mean flourescence intensity values are shown.(D) SDS-PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.(E) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 variants, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag (183-H12-5C).(F) Opti-prep purified supernatants from (E) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. Mean + SD of 5-10 experiments (each performed with virus produced from an independent transfection) is shown. (G) Virus used in (E) were used to infect SupT1 cells and productive infection was scored at 48-60 hours by immunostaining with the KC57 antibody and flow cytometry. Expression of pCMV-MxA served as a negative control. The mean + SD of 5-10 experiments is shown.(H) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP-FLAG-IFITM3 variants and immunostained with anti-FLAG M2 antibody(I) SDS-PAGE of cell lysates derived from 293T cells transfected in (H) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.(J) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 constructs, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag. Scale bar, 5 μm.(K) Opti-prep purified supernatants from (J) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. The mean + SD of 3 experiments is shown.


Figure 2 ( A ) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet - inducible pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - IFITM3 Δ1 - 21 ( Tet - ON ) immunostained with anti - FLAG M2 antibody following overnight treatment with 500 ng / mL doxycycline . Scale bar , 10 μm . ( B ) Tet - ON SupT1 cell lines were treated or not with 500 ng / mL doxycycline overnight and induction of IFITM3 protein was assessed anti - FLAG M2 immunostaining and flow cytometry . ( C ) Tet - ON SupT1 cell lines were productively infected with NL4 - 3 VSV - G and then treated with 500 ng / mL doxycycline overnight to produce virus from IFITM3 - and IFITM3 + cells . 25 ng p24 equivalents of purified were used to infect fresh Tet - ON SupT1 cells cell targets , which were previously treated with 500 ng / mL doxycycline or not . Infection was scored at 72h post - infection by immunostaining with KC57 and flow cytometry . Mean + SD of 3 experiments are shown . ( D ) 25 ng p24 equivalents of virus used in ( C ) was subjected to SDS - PAGE . Immunoblotting was performed using the anti - FLAG M2 , anti - p24 Gag , and anti - gp120 Env ( NIH # 288 ) antibodies . ( E ) 293T cells were transfected with the indicated amount of pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - Δ1 - 21 or pCMV - MxA and 1 . 0 μg of HIV - 1 pNL4 - 3 . SDS - PAGE was performed on whole cell lysates followed by immunoblotting with anti - FLAG M2 , anti - p24 Gag , anti - Env gp120 ( NIH # 288 ) , and anti - tubulin antibodies . Western blot images are representative of 3 independent experiments . Unpaired t - test , * p < 0 . 05 , ♯ p = 0 . 12 , non statistically significant . Comparisons were made between the condition indicated and the correponding condition in Tet - ON IFITM3 cells .

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

Figure 2(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm.(B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry.(C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown.(D) 25 ng p24 equivalents of virus used in (C) was subjected to SDS-PAGE. Immunoblotting was performed using the anti-FLAG M2, anti-p24 Gag, and anti-gp120 Env (NIH #288) antibodies.(E) 293T cells were transfected with the indicated amount of pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-Δ1-21 or pCMV-MxA and 1.0 μg of HIV-1 pNL4-3. SDS-PAGE was performed on whole cell lysates followed by immunoblotting with anti-FLAG M2, anti-p24 Gag, anti-Env gp120 (NIH #288), and anti-tubulin antibodies. Western blot images are representative of 3 independent experiments. Unpaired t-test, *p<0.05, ♯p=0.12, non statistically significant. Comparisons were made between the condition indicated and the correponding condition in Tet-ON IFITM3 cells.


Figure 5 ( A ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg of pQCXIP - FLAG - Human IFITM3 mutated at indicated residues and immunostained with anti - FLAG M2 antibody . Scale bars , 5 μm . The P17L , P18H , Δ22 - 24 , P18L / L23P and P18H / Δ19 - 23 mutations were introduced into the human IFITM3 background . ( B ) SDS - PAGE of FLAG - immunoprecipitated fractions from 293T cells following transfection of 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants and 0 . 5 μg of pCI - HA - NEDD4 or pCI - HA - NEDD4 C867A . Immunoblotting was performed with anti - FLAG M2 , anti - IFITM3 ( Abcam , EPR5242 ) , and an anti - ubiquitin antibody ( Enzo Life Sciences , recognizing K29 - , K48 - , and K63 - linked mono - and polyubiquitinylated proteins ) . ' L ' indicates the presence of light chain antibody derived from the FLAG antibody - coupled beads used for immunoprecipitation . * , * * , and * * * indicate mono - , di - , and tri - ubiquitinated forms of IFITM3 . ( C ) SDS - PAGE of whole cell lysate input fractions from 293T cells used in ( B ) . Immunoblotting was performed with anti - FLAG M2 , anti - NEDD4 , and anti - actin . ( D ) 293T cells were transfected with 1 . 0 μg of pNL4 - 3 and 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants . Virus - containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells . Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post - infection . The means + SD of 3 experiments are shown . ( E ) 2 . 0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV - 1NL4 - 3 in the presence of 2 μg / mL DEAE - Dextran . Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post - infection . The ' Colobus native ' sequence represents the full IFITM3 sequence identified in the colobus monkey . ( F ) As in ( E ) , except cells were challenged with Influenza A virus H1N1 PR / 8 / 34 , ( Charles River Laboratories ) , at a dose that resulted in ˜ 50 % infection of control ( Empty ) cells ( equivalent to 103 TCID50 / 0 . 2 mL ) . Infection was scored by immunostaining with an anti - IAV NP antibody and flow cytometry at 18 hours post - infection . Results are presented in a logarithmic scale . Means + SD of 3 - 5 experiments are shown . Unpaired t - test , * p < 0 . 05 , * * p < 0 . 005 .

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

Figure 5(A) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg of pQCXIP-FLAG-Human IFITM3 mutated at indicated residues and immunostained with anti-FLAG M2 antibody. Scale bars, 5 μm. The P17L, P18H, Δ22-24, P18L/L23P and P18H/Δ19-23 mutations were introduced into the human IFITM3 background.(B) SDS-PAGE of FLAG-immunoprecipitated fractions from 293T cells following transfection of 0.5 μg of pQCXIP-FLAG-IFITM3 variants and 0.5 μg of pCI-HA-NEDD4 or pCI-HA-NEDD4 C867A. Immunoblotting was performed with anti-FLAG M2, anti-IFITM3 (Abcam, EPR5242), and an anti-ubiquitin antibody (Enzo Life Sciences, recognizing K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins). 'L' indicates the presence of light chain antibody derived from the FLAG antibody-coupled beads used for immunoprecipitation. *, **, and *** indicate mono-, di-, and tri-ubiquitinated forms of IFITM3.(C) SDS-PAGE of whole cell lysate input fractions from 293T cells used in (B). Immunoblotting was performed with anti-FLAG M2, anti-NEDD4, and anti-actin.(D) 293T cells were transfected with 1.0 μg of pNL4-3 and 0.5 μg of pQCXIP-FLAG-IFITM3 variants. Virus-containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells. Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post-infection. The means + SD of 3 experiments are shown.(E) 2.0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV-1NL4-3 in the presence of 2 μg/mL DEAE-Dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post-infection. The 'Colobus native' sequence represents the full IFITM3 sequence identified in the colobus monkey. (F) As in (E), except cells were challenged with Influenza A virus H1N1 PR/8/34, (Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 103 TCID50 / 0.2 mL). Infection was scored by immunostaining with an anti-IAV NP antibody and flow cytometry at 18 hours post-infection. Results are presented in a logarithmic scale. Means + SD of 3-5 experiments are shown. Unpaired t-test, *p<0.05, **p<0.005.


Figure 1Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK - p25 animal mice . Colors indicate fold change relative to control animals on a log2 - scale . Row colors ( left ) indicate peptides from predicted cell - type specific proteins . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte . Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice . Colors indicate fold change relative to control animals on a log2 - scale . Row colors ( left ) indicate peptides from predicted cell - type specific proteins . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte . Colors indicate fold change relative to control animals on a log2 - scale . Row colors ( left ) indicate peptides from predicted cell - type specific proteins . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte . ( E ) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals . ( F - G ) Relative abundances across all tissues for phosphotyrosine peptides : ( F ) SVyTEIK ( Siglec - F pY561 ) ; ( G ) LyDFVK ( Inpp5d pY868 ) . Bars indicate mean ± 95 % confidence interval ( CI ) ; n = 3 - 8 samples ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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 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

Figure 1Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK-p25 animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte.  Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. (E) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals. (F-G) Relative abundances across all tissues for phosphotyrosine peptides: (F) SVyTEIK (Siglec-F pY561); (G) LyDFVK (Inpp5d pY868). Bars indicate mean ± 95% confidence interval (CI); n = 3-8 samples; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


Figure 2 ( A ) Immunofluorescence ( IF ) staining showing Siglec - F and Iba1 localization in hippocampus CA3 region of 3wk induced CK ( top ) or CK - p25 ( bottom ) mice . Scale bars = 50 μm . Colors are : Magenta = Iba1 , Cyan = Siglec - F . Images are 60x super - resolution max z - stack projections taken from coronal slices . ( B - C ) Percent area coverage of ( B ) Iba1 and ( C ) Iba1 - proximal Siglec - F between CK and CK - p25 mice across hippocampal regions . Bars indicate mean ± 95 % CI ; n = 3 - 4 animals ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . ( D - E ) IF staining of Siglec - F and Iba1 in ( D ) 5XFAD 2 month hypothalamus ( HY ) and ( E ) 5XFAD 6 month dentate gyrus ( DG ) . Yellow = Aβ from methoxy X04 ( D ) or D54D2 ( E ) . Scale bars = 50 μm . ( F ) Percent area coverage of Iba1 - proximal Siglec - F relative to total Iba1 across all analyzed thalamic , cortical , and hippocampal regions of 6 month WT ( non - Tg ) and 5XFAD mice . Box indicates quartiles and whiskers indicate the last datum within 1 . 5 inter - quartile range ; n = 15 - 40 images ( 3 - 5 animals ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . ( G ) Percent area coverage of Siglec - F localized to Iba1 compared to total Aβ plaque volume in each field of view across all analyzed images from 5XFAD and non - Tg control mice . Linear regression R2 value is shown for images with ≥ 10 μm2 total amyloid volume . ( H ) Cumulative distribution function ( CDF ) of the distance between Siglec - F + and Iba1 + mask voxels and nearest Aβ plaques in 6 month 5XFAD mice . Each value in the CDF represents the mean distance calculated from a single z - stack of a ROI containing ≥ 1 plaque of size ≥ 10 μm2 . n = 55 images ( 4 animals ) . ( I - J ) Relative RNA abundances for Siglec - 8 in ( H ) hippocampus and ( I ) posterior cingulate cortex post - mortem patient tissue . Linear regression R2 values are shown on plots . ( K ) IF staining showing Siglec - 8 and Iba1 , and MHC - II in cortical white matter tissue from a patient with late - onset AD ( 20x wide - field ; Case ID : 01 - 43 ) . Scale bars = 50 μm . Images are 60x super - resolution max z - stack projections . ( L - M ) Percent area coverage of Siglec - 8 localized to Iba1 or MHC in individuals with non - AD or AD . In ( K ) each point represents one WM or GM region average from each individual . In ( L ) values and statistics are shown for each image analyzed . Boolean mask operators indicate : ° = mask dilation , ⋂ = intersection , ⋃ = union , NOT = inverse mask . Box indicates quartiles and whiskers indicate the last datum within 1 . 5 inter - quartile range ; n = 8 - 43 images ( n = 1 ND , 3 early - onset AD , 4 late - onset AD ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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

Figure 2(A) Immunofluorescence (IF) staining showing Siglec-F and Iba1 localization in hippocampus CA3 region of 3wk induced CK (top) or CK-p25 (bottom) mice. Scale bars = 50 μm. Colors are: Magenta = Iba1, Cyan = Siglec-F. Images are 60x super-resolution max z-stack projections taken from coronal slices. (B-C) Percent area coverage of (B) Iba1 and (C) Iba1-proximal Siglec-F between CK and CK-p25 mice across hippocampal regions. Bars indicate mean ± 95% CI; n = 3-4 animals; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (D-E) IF staining of Siglec-F and Iba1 in (D) 5XFAD 2 month hypothalamus (HY) and (E) 5XFAD 6 month dentate gyrus (DG). Yellow = Aβ from methoxy X04 (D) or D54D2 (E). Scale bars = 50 μm. (F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (G) Percent area coverage of Siglec-F localized to Iba1 compared to total Aβ plaque volume in each field of view across all analyzed images from 5XFAD and non-Tg control mice. Linear regression R2 value is shown for images with ≥10 μm2 total amyloid volume.  (H) Cumulative distribution function (CDF) of the distance between Siglec-F+ and Iba1+ mask voxels and nearest Aβ plaques in 6 month 5XFAD mice. Each value in the CDF represents the mean distance calculated from a single z-stack of a ROI containing ≥ 1 plaque of size ≥10 μm2. n = 55 images (4 animals). (I-J) Relative RNA abundances for Siglec-8 in (H) hippocampus and (I) posterior cingulate cortex post-mortem patient tissue. Linear regression R2 values are shown on plots.(K) IF staining showing Siglec-8 and Iba1, and MHC-II in cortical white matter tissue from a patient with late-onset AD (20x wide-field; Case ID: 01-43). Scale bars = 50 μm. Images are 60x super-resolution max z-stack projections.(L-M) Percent area coverage of Siglec-8 localized to Iba1 or MHC in individuals with non-AD or AD. In (K) each point represents one WM or GM region average from each individual. In (L) values and statistics are shown for each image analyzed. Boolean mask operators indicate: ° = mask dilation, ⋂ = intersection, ⋃ = union, NOT = inverse mask. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 8-43 images (n = 1 ND, 3 early-onset AD, 4 late-onset AD); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


Figure 3 ( A ) Flow cytometry quantification of BV - 2 Siglec - F expression levels . Cells were cotreated with 0 - 10 ng / mL IFNγ and 0 - 10 μM tofacitinib . Values are geometric mean fluorescence intensities ( gMFI ) . Bars indicate mean ± 95 % CI ; n = 2 - 4 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated group . ( B ) IF staining of iMGLs treated with IFNγ . Siglec - 8 fluorescence is only shown on non - nuclear ( Hoechst 33342 - ) regions . Images are 60x super - resolution max z - stack projections . Colors are : Red = Iba1 , Green = Siglec - 8 , Blue = 33342 . Scale bars = 20 μm . ( C ) Quantification of Siglec - 8 on iMGLs treated with PBS or 25 ng / mL IFNα , IFNβ , or IFNγ . Values are mean Siglec - 8 intensity values from Iba1 + ; 33342 - regions . Bars indicate mean ± 95 % CI ; n = 8 images ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated group . ( D ) Quantification of Siglec - 8 on iMGLs treated ± 25 ng / mL IFNγ and 5 μM tofacitinib . Legend is same as ( C ) . Bars indicate mean ± 95 % CI ; n = 5 - 20 images ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated group .

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

Figure 3(A) Flow cytometry quantification of BV-2 Siglec-F expression levels. Cells were cotreated with 0-10 ng/mL IFNγ and 0-10 μM tofacitinib. Values are geometric mean fluorescence intensities (gMFI). Bars indicate mean ± 95% CI; n = 2-4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group. (B) IF staining of iMGLs treated with IFNγ. Siglec-8 fluorescence is only shown on non-nuclear (Hoechst 33342-) regions. Images are 60x super-resolution max z-stack projections. Colors are: Red = Iba1, Green = Siglec-8, Blue = 33342. Scale bars = 20 μm. (C) Quantification of Siglec-8 on iMGLs treated with PBS or 25 ng/mL IFNα, IFNβ, or IFNγ. Values are mean Siglec-8 intensity values from Iba1+;33342- regions. Bars indicate mean ± 95% CI; n = 8 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.(D) Quantification of Siglec-8 on iMGLs treated ± 25 ng/mL IFNγ and 5 μM tofacitinib. Legend is same as (C). Bars indicate mean ± 95% CI; n = 5-20 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.


Figure 4 ( A ) Confluency doubling times quantified from BV - 2 cells with stable retroviral expression of Siglec - F and related human Siglec constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field images . Cells grown in media alone or + 50 mU sialidase ( + SA ) . Bars indicate mean ± 95 % CI ; n = 3 - 12 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F groups . ( B ) Flow cytometry quantification of cell live / dead markers Annexin - V and propidium iodide on BV - 2 cells with stable retrovirus expression of Siglec - F constructs . % parent values are shown for early apoptotic ( Annexin - V + ; PI ‑ ) , late apoptotic ( Annexin - V + ; PI ‑ ) , and necrotic ( Annexin - V - ; PI + ) populations . Bars indicate mean ± SD ; n = 8 replicates . ( C ) Confluency doubling times quantified from BV - 2 cells with dox - inducible expression of Siglec - F constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field images . Bars indicate mean ± SD ; n = 8 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group . ( D ) Selected gene expression changes ( top ) and enriched gene sets ( bottom ) taken from RNA - seq analysis of BV - 2 cells with stable retroviral expression of Siglec constructs , 2xY - > F , and empty vector controls . Heatmaps share a colormap range of 0 . 25 to 4 for z - score values ( top ) , normalized enrichment scores ( NES , bottom ) . ( F ) Expression levels of C1qa , IL - 1β , and β - actin by qPCR analysis in BV - 2 cells with inducible Siglec - F expression after 48 hours of treatment with 500 ng / mL doxycycline . Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group . ( G ) Western blot showing pro - IL - 1β and β - tubulin in BV - 2 cells induced to express Siglec - F at 0 and 48 hours .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 4 (A) Confluency doubling times quantified from BV-2 cells with stable retroviral expression of Siglec-F and related human Siglec constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Cells grown in media alone or + 50 mU sialidase (+SA). Bars indicate mean ± 95% CI; n = 3-12 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F groups. (B) Flow cytometry quantification of cell live / dead markers Annexin-V and propidium iodide on BV-2 cells with stable retrovirus expression of Siglec-F constructs. % parent values are shown for early apoptotic (Annexin-V+;PI‑), late apoptotic (Annexin-V+;PI‑), and necrotic (Annexin-V-;PI+) populations. Bars indicate mean ± SD; n = 8 replicates. (C) Confluency doubling times quantified from BV-2 cells with dox-inducible expression of Siglec-F constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Bars indicate mean ± SD; n = 8 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. (D) Selected gene expression changes (top) and enriched gene sets (bottom) taken from RNA-seq analysis of BV-2 cells with stable retroviral expression of Siglec constructs, 2xY->F, and empty vector controls. Heatmaps share a colormap range of 0.25 to 4 for z-score values (top), normalized enrichment scores (NES, bottom).(F) Expression levels of C1qa, IL-1β, and β-actin by qPCR analysis in BV-2 cells with inducible Siglec-F expression after 48 hours of treatment with 500 ng/mL doxycycline. Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.(G) Western blot showing pro-IL-1β and β-tubulin in BV-2 cells induced to express Siglec-F at 0 and 48 hours.


Figure 5 ( B - C ) Volcano plots showing phosphotyrosine peptides that were ( B ) increased or ( C ) decreased after Siglec - F expression . Plots show Siglec - F compared to 2xY - > F mutant data integrated across all three runs . Protein names are shown for altered peptides . Labels are only shown for peptides with maximum directional change from each protein . Labels colored according to GO terms : magenta = signal transduction , red = endosome , cyan = cell adhesion , yellow = cytoskeleton , green = metabolism . ( D ) Heatmap showing phosphosites in CK - p25 which overlapped in their directional change with BV - 2 Siglec - F - associated sites . Colors indicate fold change relative to 2xY - > F controls . Heatmap showing phosphosites from CD33 , Siglec - 5 , and Siglec - 8 overexpression that were increased with Siglec expression and shared their directional change with Siglec - F . Siglec - F column indicates average of all untreated Siglec - F replicates . Color scale is same as ( D ) . Heatmap showing phosphosites from CD33 , Siglec - 5 , and Siglec - 8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec - F . Siglec - F column indicates average of all untreated Siglec - F replicates . Color scale is same as ( D ) . ( G ) Western blot quantification of myc , SHP - 2 , and SHP - 1 from myc co - IP eluates . Co - IP lysates were prepared from BV - 2 cells stably expressing Siglec - F and optionally treated with 1 μM SHP099 . ( H ) Heatmap showing phosphosites that were perturbed by SHP099 treatment . Color scale is same as ( D ) .

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

Figure 5(B-C) Volcano plots showing phosphotyrosine peptides that were (B) increased or (C) decreased after Siglec-F expression. Plots show Siglec-F compared to 2xY->F mutant data integrated across all three runs. Protein names are shown for altered peptides. Labels are only shown for peptides with maximum directional change from each protein. Labels colored according to GO terms: magenta = signal transduction, red = endosome, cyan = cell adhesion, yellow = cytoskeleton, green = metabolism. (D) Heatmap showing phosphosites in CK-p25 which overlapped in their directional change with BV-2 Siglec-F-associated sites. Colors indicate fold change relative to 2xY->F controls.  Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were increased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).  Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D). (G) Western blot quantification of myc, SHP-2, and SHP-1 from myc co-IP eluates. Co-IP lysates were prepared from BV-2 cells stably expressing Siglec-F and optionally treated with 1 μM SHP099.(H) Heatmap showing phosphosites that were perturbed by SHP099 treatment. Color scale is same as (D).