Figure 1A Mass spectrometric analysis of LSD1 methylation . Immunoprecipitation ( IP ) was performed using anti - Flag antibody in HEK - 293T cells overexpressing Flag - tagged LSD1 , followed by liquid chromatography coupled tandem mass spectrometry ( LC - MS / MS ) analysis . The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838 . B Interactions between LSD1 and CARM1 were detected by co - immunoprecipitation ( Co - IP ) assay with anti - Flag in HEK - 293T cells transfected with Flag - LSD1 or Flag - CARM1 , respectively . C Interaction of endogenous LSD1 with CARM1 was analyzed by Co - IP assay using anti - CARM1 antibody in MDA - MB - 231 cells . Purified GST - tagged CARM1 was pulled down with the Flag - LSD1 purified from HEK - 293T cells . The amounts of GST and GST - tagged CARM1 were visualized by Coomassie Blue staining ( asterisk : GST - CARM1 ) . E In vitro methylation assays . Purified fusion protein of GST - tagged LSD1 WT or the four methylated arginine mutants ( R - A ) , or four methylated arginine mutants in combination ( 4RA ) , were incubated with GST - CARM1 in the presence of 3H - SAM . Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining ( arrows : the position of LSD1 ; arrowheads : the position of CARM1 ) . F Increasing amounts of GST - CARM1 incubated with GST - LSD1 in presence of SAM at 30 ° C for 1 h . And methylation of LSD1 was analyzed by immunoblotting with anti - LSD1 R838me2a antibody . G GST - CARM1 incubated with GST - LSD1 WT or GST - LSD1 R838A / R838K in presence of SAM at 30 ° C for 1 h , followed by immunoblotting with anti - LSD1 R838me2a to detect LSD1 methylation .

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Figure 1A Mass spectrometric analysis of LSD1 methylation. Immunoprecipitation (IP) was performed using anti-Flag antibody in HEK-293T cells overexpressing Flag-tagged LSD1, followed by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838.B Interactions between LSD1 and CARM1 were detected by co-immunoprecipitation (Co-IP) assay with anti-Flag in HEK-293T cells transfected with Flag-LSD1 or Flag-CARM1, respectively.C Interaction of endogenous LSD1 with CARM1 was analyzed by Co-IP assay using anti-CARM1 antibody in MDA-MB-231 cells.Purified GST-tagged CARM1 was pulled down with the Flag-LSD1 purified from HEK-293T cells. The amounts of GST and GST-tagged CARM1 were visualized by Coomassie Blue staining (asterisk: GST-CARM1).E In vitro methylation assays. Purified fusion protein of GST-tagged LSD1 WT or the four methylated arginine mutants (R-A), or four methylated arginine mutants in combination (4RA), were incubated with GST-CARM1 in the presence of 3H-SAM. Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining (arrows: the position of LSD1; arrowheads: the position of CARM1).F Increasing amounts of GST-CARM1 incubated with GST-LSD1 in presence of SAM at 30°C for 1 h. And methylation of LSD1 was analyzed by immunoblotting with anti-LSD1 R838me2a antibody.G GST-CARM1 incubated with GST-LSD1 WT or GST-LSD1 R838A/R838K in presence of SAM at 30°C for 1 h, followed by immunoblotting with anti-LSD1 R838me2a to detect LSD1 methylation.


Figure 2A Methylation of LSD1 was assessed in MCF10A , MCF7 and MDA - MB - 231 cells by immunoblotting using anti - LSD1 R838me2a . Protein expression levels of LSD1 in MDA - MB - 231 cells transfected with CARM1 shRNAs or control vector , were assessed by western blotting ( B ) ( Error bars represent the mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - test ) . relative mRNA expression levels of LSD1 in MDA - MB - 231 cells transfected with CARM1 shRNAs or control vector , were assessed by qPCR analysis ( C ) ( Error bars represent the mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - test ) . D MDA - MB - 231 cells were treated with the indicated amounts of MS049 for 48 h , LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies . E MDA - MB - 231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 ( 10 μM ) or DMSO , before LSD1 level was analyzed by immunoblotting . F MDA - MB - 231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h , and the cell lysates were analyzed by western blotting . G MDA - MB - 231 cells were transfected with CARM1 shRNA # 2 or control vector , and then treated with CHX ( 100 μg / ml ) for the indicated time ; LSD1 protein level was examined by western blotting . H MDA - MB - 231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h . Cell lysates were immunoprecipitated by anti - LSD1 antibody and then analysed by immunoblotting . I MDA - MB - 231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h . The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti - LSD1 antibody . J HEK - 293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time . LSD1 were examined by western blotting analysis . K HEK - 293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h . Cell lysates were immunoprecipitated using anti - Flag antibody and then subjected to immunoblotting . L HEK - 293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h . Co - IP assay was performed using anti - Flag and then subjected to western blotting .

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Figure 2A Methylation of LSD1 was assessed in MCF10A, MCF7 and MDA-MB-231 cells by immunoblotting using anti-LSD1 R838me2a.Protein expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by western blotting (B) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).relative mRNA expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by qPCR analysis (C) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).D MDA-MB-231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies.E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.F MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by western blotting.G MDA-MB-231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by western blotting.H MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-LSD1 antibody and then analysed by immunoblotting.I MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti-LSD1 antibody.J HEK-293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time. LSD1 were examined by western blotting analysis.K HEK-293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-Flag antibody and then subjected to immunoblotting.L HEK-293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co-IP assay was performed using anti-Flag and then subjected to western blotting.


Figure 3A Immunoprecipitation assay was performed using anti - Flag antibody in MM - 231 - shLSD1 - WT / R838A cells . The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies . B MDA - MB - 231 cells were treated with the indicated amounts of P5091 for 48 h , LSD1 protein levels were assessed by immunoblotting . C MDA - MB - 231 cells were treated with P5091 ( 10 μM ) for 48 h and MG132 for 10 h . The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti - LSD1 antibody . D HEK - 293T cells were transfected with USP7 WT or USP7 C223S , then treated with MG132 for 10 h . Cell lysates were immunoprecipitated using anti - LSD1 antibody followed by immunoblotting analysis . E MDA - MB - 231 cells overexpressing Flag - tagged LSD1 WT / R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h . Cell lysates were immunoprecipitated by anti - Flag antibody and then analysed by immunoblotting . F MDA - MB - 231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h . And cell lysates were immunoprecipitated with anti - LSD1 then subjected to immunoblotting . G MDA - MB - 231 cells were treated with or without MS049 for 48 h and MG132 for 10 h , Cell lysates were immunoprecipitated using anti - LSD1 antibody and then analysed by immunoblotting . H HEK - 293T cells were transfected with USP7 WT or USP7 C223S , then treated with or without MS049 for 48 h and MG132 for 10 h . Cell lysates were immunoprecipitated with anti - LSD1 antibody followed by immunoblotting analysis .

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Figure 3A Immunoprecipitation assay was performed using anti-Flag antibody in MM-231-shLSD1-WT/R838A cells. The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies.B MDA-MB-231 cells were treated with the indicated amounts of P5091 for 48 h, LSD1 protein levels were assessed by immunoblotting.C MDA-MB-231 cells were treated with P5091 (10 μM) for 48 h and MG132 for 10 h. The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti-LSD1 antibody.D HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-LSD1 antibody followed by immunoblotting analysis.E MDA-MB-231 cells overexpressing Flag-tagged LSD1 WT/R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated by anti-Flag antibody and then analysed by immunoblotting.F MDA-MB-231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h. And cell lysates were immunoprecipitated with anti-LSD1 then subjected to immunoblotting.G MDA-MB-231 cells were treated with or without MS049 for 48 h and MG132 for 10 h, Cell lysates were immunoprecipitated using anti-LSD1 antibody and then analysed by immunoblotting.H HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with or without MS049 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated with anti-LSD1 antibody followed by immunoblotting analysis.


Figure 4Migration assays ( 24 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( A ) with or without MS049 treatment . Representative micrographs of migrated cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars indicate the mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . invasion assays ( 48 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( B ) with or without MS049 treatment . Representative micrographs of invaded cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . Migration assays ( 24 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F cells ( C ) with or without MS049 treatment . Representative micrographs of migrated cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars indicate the mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . D invasion assays ( 48 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F cells ( D ) with or without MS049 treatment . Representative micrographs of invaded cells are shown . Data represent the number of cells derived from mean cell counts of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - test ) . E MM - 231 - shLSD1 - WT / R838A / R838K / R838F cells ( 1 × 106 ) were injected into the tail veins of 5 - week - old female nude mice , and 7 weeks later , mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions . The number of visible surface metastatic lesions in lungs was counted ( mean of 7 mice ) ( Data are mean ± SD , n = 7 mice for each group , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . F Lung metastatic nodules confirmed by haematoxylin and eosin staining . Scale bars , 100 μm .

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Figure 4Migration assays (24 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (A) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).invasion assays (48 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (B) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).Migration assays (24 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (C) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).D invasion assays (48 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (D) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).E MM-231-shLSD1-WT/R838A/R838K/R838F cells (1×106) were injected into the tail veins of 5-week-old female nude mice, and 7 weeks later, mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions. The number of visible surface metastatic lesions in lungs was counted (mean of 7 mice) (Data are mean ± SD, n = 7 mice for each group, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.


Figure 5MDA - MB - 231 cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E - cadherin ( A ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test ) . B MDA - MB - 231 cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin ( B ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test ) . MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 , real - time PCR was performed to analysis the E - cadherin ( C ) mRNA levels ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . D MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 , real - time PCR was performed to analysis the vimentin ( D ) mRNA levels ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E - cadherin ( E ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) . F MM - 231 - shLSD1 - WT / R838A cells were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin ( F ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - test ) .

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Figure 5MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (A) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).B MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (B) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the E-cadherin (C) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).D MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091, real-time PCR was performed to analysis the vimentin (D) mRNA levels (Data are are presented as mean ± SD, n = 3 experimental replicates, **P < 0.01, ns = not significant, Student's t-test).MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (E) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).F MM-231-shLSD1-WT/R838A cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 or anti-H3K4me2 or anti-H3K9me2 antibody, and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (F) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).


Figure 6A Typical pictures of the immunohistochemical staining of LSD1 R838me2a , LSD1 and CARM1 in breast cancer samples . Scale bars , 100 μm . The percentage of samples with high level of LSD1 R838me2a ( B ) expression in different TNM stages of breast cancer ( n = 70 breast tumor samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 test ) . C , D The percentage of samples with high level of , LSD1 ( C ) and CARM1 ( D ) expression in different TNM stages of breast cancer ( n = 70 breast tumor samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 test ) . The correlation of LSD1 R838me2a with LSD1 ( E ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al . F , G The correlation of , CARM1 with LSD1 R838me2a ( F ) and CARM1 with LSD1 ( G ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al .

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Figure 6A Typical pictures of the immunohistochemical staining of LSD1 R838me2a, LSD1 and CARM1 in breast cancer samples. Scale bars, 100 μm.The percentage of samples with high level of LSD1 R838me2a (B) expression in different TNM stages of breast cancer (n = 70 breast tumor samples; B, P = 0.0095; C, P = 0.002; D, P = 0.0024; χ2 test).C, D The percentage of samples with high level of , LSD1 (C) and CARM1 (D) expression in different TNM stages of breast cancer (n = 70 breast tumor samples; B, P = 0.0095; C, P = 0.002; D, P = 0.0024; χ2 test).The correlation of LSD1 R838me2a with LSD1 (E) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.F, G The correlation of , CARM1 with LSD1 R838me2a (F) and CARM1 with LSD1 (G) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.


Figure 1A . Volcano blot representation of the differently expressed genes in FCRLS - and CD11b - positive Grn - / - microglia in comparison to wt microglia isolated from 5 . 5 - month - old mice ( male , n = 5 ) . 116 genes out of 529 genes analyzed , are significantly changed , and from these 58 genes are up - and 58 genes are down regulated . Eight genes with the highest fold change are highlighted . B . Heatmap of significantly affected genes ( p < 0 . 05 ) in FCRLS - and CD11b - positive Grn - / - microglia in comparison to Grn + / + microglia isolated from 5 . 5 - month - old mice ( n = 5 per genotype ) . For the significantly affected genes of the Grn - / - microglia , mRNA expression data of the Trem2 - / - microglia in comparison to the corresponding wt microglia were taken from previously published data ( Mazaheri et al , 2017 ) . The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation ( n ≥ 5 per genotype ) . * labelled genes were not analyzed or below detection limit in Trem2 - / - microglia mRNA expression data set ( Mazaheri et al , 2017 ) . D . Gene expression levels of FCRLS - and CD11b - positive Grn - / - microglia normalized to wt in comparison to the published normalized data set of Trem2 - / - microglia ( Mazaheri et al , 2017 ) .

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 O O O O O O O O O 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

Figure 1A. Volcano blot representation of the differently expressed genes in FCRLS- and CD11b- positive Grn-/- microglia in comparison to wt microglia isolated from 5.5-month-old mice (male, n = 5). 116 genes out of 529 genes analyzed, are significantly changed, and from these 58 genes are up- and 58 genes are down regulated. Eight genes with the highest fold change are highlighted.B. Heatmap of significantly affected genes (p < 0.05) in FCRLS- and CD11b-positive Grn-/- microglia in comparison to Grn+/+ microglia isolated from 5.5-month-old mice (n = 5 per genotype). For the significantly affected genes of the Grn-/- microglia, mRNA expression data of the Trem2-/- microglia in comparison to the corresponding wt microglia were taken from previously published data (Mazaheri et al, 2017). The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation (n ≥ 5 per genotype). * labelled genes were not analyzed or below detection limit in Trem2-/- microglia mRNA expression data set (Mazaheri et al, 2017).D. Gene expression levels of FCRLS- and CD11b-positive Grn-/- microglia normalized to wt in comparison to the published normalized data set of Trem2-/- microglia (Mazaheri et al, 2017).


Figure 2A . Immunoblot analysis of ApoE , CLEC7A , TREM2 and CD68 in lysates of acutely isolated microglia from Grn - / - and Grn + / + mice ( 9 months of age , n = 3 per genotype , female ) . IBA1 was used as loading control . B . Quantification of protein expression normalized to levels of Grn + / + microglia ( n = 3 ) . Data represent the mean ± SD . C . ELISA mediated quantification of sTREM2 in brain homogenates of Grn - / - and Grn + / + mice Data are shown as mean ± SEM ( n = 3 - 6 ) . D . ELISA mediated quantification of sTREM2 in serum of Grn - / - and Grn + / + mice . Data are shown as mean ± SEM ( n = 4 - 13 ) . E . Microglial expression of P2RY12 in cortical sections of wt , Grn - / - and Trem2 - / - mice . ( 9 months of age , female ) F . Quantification of P2RY12 positive microglia . Data are shown as mean ± SD ( n = 3 per genotype , female , except one male for Trem2 - / - ) . G . Immunoblot analysis of secreted PGRN and ApoE ( ApoEmed ) in conditioned media and PGRN , ApoE , CLEC7A , P2RY12 and TREM2 in lysates of BV2 wild type ( Grnwt ) and PGRN deficient ( Grnmut ) cells . Soluble amyloid precursor protein ( APPs ) and calnexin were used as a loading control . H . Quantification of Immunoblots normalized to BV2 Grnwt levels ( n = 3 - 5 ) . Data represent the mean ± SD . I . ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells . Data represent the mean ± SDJ . Quantification of relative mRNA levels of Apoe , Clec7a , P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells . Data represent the mean ± SD .

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

Figure 2A. Immunoblot analysis of ApoE, CLEC7A, TREM2 and CD68 in lysates of acutely isolated microglia from Grn-/- and Grn+/+ mice (9 months of age, n=3 per genotype, female). IBA1 was used as loading control.B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n=3). Data represent the mean ± SD.C. ELISA mediated quantification of sTREM2 in brain homogenates of Grn-/- and Grn+/+ mice Data are shown as mean ± SEM (n=3-6). D. ELISA mediated quantification of sTREM2 in serum of Grn-/- and Grn+/+ mice. Data are shown as mean ± SEM (n=4-13). E. Microglial expression of P2RY12 in cortical sections of wt, Grn-/-and Trem2-/- mice. (9 months of age, female) F. Quantification of P2RY12 positive microglia. Data are shown as mean ± SD (n=3 per genotype, female, except one male for Trem2-/-). G. Immunoblot analysis of secreted PGRN and ApoE (ApoEmed) in conditioned media and PGRN, ApoE, CLEC7A, P2RY12 and TREM2 in lysates of BV2 wild type (Grnwt) and PGRN deficient (Grnmut) cells. Soluble amyloid precursor protein (APPs) and calnexin were used as a loading control.H. Quantification of Immunoblots normalized to BV2 Grnwt levels (n=3-5). Data represent the mean ± SD.I. ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SDJ. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD.


Figure 3A . Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E . coli as target particles . Phagocytosis was terminated after 30 and 60 minutes ( min ) of incubation . Data are presented as mean percentage of cells positive for pHrodo uptake ± S . D ( n = 3 ) . B . Enhanced migration of CD68 positive , PGRN deficient cells in an ex vivo model . Immunofluorescence analysis of cultured Grn + / + and Grn - / - organotypic brain at 7 DIV immunostained using microglial marker CD68 . Nuclei were counterstained using DAPI . Images of boxed regions in left panels are shown at higher magnifications in right panels . Scale bars : 500 µm ( left panels ) , 250 µm ( right panels ) . Quantitative analysis reveals that Grn - / - microglia migrate larger distances than Grn + / + . Data are shown as mean ± SD ( n = 3 independent experiments ) Quantification displays an increased number of migrating Grn - / - cells compared to Grn + / + cells . Data are shown as mean ± SD ( n = 3 independent experiments ) E . Enhanced microglial clustering around amyloid plaques in the absence of PGRN . Left : IBA1 - stained microglia clustering around X - 34 - positive amyloid plaque cores in 4 - month - old APPPS1 / Grn + / + mice . Right : Age - matched APPPS1 / Grn - / - mice display microglial hyper - clustering around amyloid plaques compared to APPPS1 / Grn + / + . Dotted white boxes indicate the area that is shown at higher magnification . Scale bars indicate 10µm and 3µm in inset . Immunohistochemical quantification of IBA1 positive microglial cell number per

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

Figure 3A. Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 minutes (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± S.D (n=3).B. Enhanced migration of CD68 positive, PGRN deficient cells in an ex vivo model. Immunofluorescence analysis of cultured Grn+/+ and Grn-/- organotypic brain at 7 DIV immunostained using microglial marker CD68. Nuclei were counterstained using DAPI. Images of boxed regions in left panels are shown at higher magnifications in right panels. Scale bars: 500 µm (left panels), 250 µm (right panels). Quantitative analysis reveals that Grn-/- microglia migrate larger distances than Grn+/+. Data are shown as mean ± SD (n=3 independent experiments) Quantification displays an increased number of migrating Grn-/- cells compared to Grn+/+ cells. Data are shown as mean ± SD (n=3 independent experiments)E. Enhanced microglial clustering around amyloid plaques in the absence of PGRN. Left: IBA1-stained microglia clustering around X-34-positive amyloid plaque cores in 4-month-old APPPS1/Grn+/+ mice. Right: Age-matched APPPS1/Grn-/- mice display microglial hyper-clustering around amyloid plaques compared to APPPS1/Grn+/+. Dotted white boxes indicate the area that is shown at higher magnification. Scale bars indicate 10µm and 3µm in inset. Immunohistochemical quantification of IBA1 positive microglial cell number per plaqu


Figure 4A . TSPO - µPET in rodents : Axial slices ( A1 , A2 , A3 ) of averaged % - TSPO - PET differences between Grn - / - or Trem2 - / - mice and wt indicate increased microglial activity in the cerebrum of Grn - / - mice ( hot color scale ) and decreased microglial activity in the cerebrum of Trem2 - / - mice ( cold color scale ) . Scatter plot depicts single TSPO - µPET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 8 at 11 months ; wt n = 10 at 8 - 11 months ) . Statistics derive from one - way ANOVA with Tukey post - hoc test . B . PGRN serum levels of the patients subjected to TSPO - PET confirmed the GRN mutation and non - GRN mutation carrier . PGRN was measured by ELISA and normalized to serum levels of a healthy control . C . TSPO - PET in human FTLD patients : Visual interpretation of axial TSPO - PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices ( white arrows ) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation . PET results are illustrated upon an MRI template ; 60 - 80 min p . i . emission recording ; global mean scaling . Both patients were low - affinity binder .

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 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 O O O O O O O O O O O

Figure 4A. TSPO-µPET in rodents: Axial slices (A1, A2, A3) of averaged %-TSPO-PET differences between Grn-/-or Trem2-/- mice and wt indicate increased microglial activity in the cerebrum of Grn-/- mice (hot color scale) and decreased microglial activity in the cerebrum of Trem2-/- mice (cold color scale). Scatter plot depicts single TSPO-µPET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=8 at 11 months; wt n=10 at 8-11 months). Statistics derive from one-way ANOVA with Tukey post-hoc test.B. PGRN serum levels of the patients subjected to TSPO-PET confirmed the GRN mutation and non-GRN mutation carrier. PGRN was measured by ELISA and normalized to serum levels of a healthy control.C. TSPO-PET in human FTLD patients: Visual interpretation of axial TSPO-PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices (white arrows) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation. PET results are illustrated upon an MRI template; 60-80 min p.i. emission recording; global mean scaling. Both patients were low-affinity binder.


Figure 5FDG - µPET in rodents : Axial slices ( as indicated in Fig . 4A ) of averaged % - FDG - µPET differences between Grn - / - or Trem2 - / - mice and wt indicate decreased glucose metabolism in the cerebrum of Grn - / - and Trem2 - / - ( both cold color scale ) when compared to wt . Scatter plot depicts single FDG - PET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 6 at 11 months ; wt n = 10 at 8 - 11 months ) .

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

Figure 5FDG-µPET in rodents: Axial slices (as indicated in Fig. 4A) of averaged %-FDG-µPET differences between Grn-/-or Trem2-/- mice and wt indicate decreased glucose metabolism in the cerebrum of Grn-/- and Trem2-/- (both cold color scale) when compared to wt. Scatter plot depicts single FDG-PET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=6 at 11 months; wt n=10 at 8-11 months).


Figure 1A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( A ) : 200 μm . A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell migration ( B ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( B ) : 100 μm . ( C ) Effect of WDR63 knockdown on the migration of A549 cells was analyzed by single cell tracking with time - lapse microscopy at 10 min intervals for 4 h . Scale bar : 200 μm . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . ( D ) A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell invasion assay . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm . A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell migration ( E ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm . A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to single cell tracking ( F ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar 200 μm . A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell invasion ( G ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm . ( H ) A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 2 ( each also expressing luciferase ) were injected via tail vein into nude mice ( n = 6 for each group ) . Three weeks after injection , tumor formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

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

Figure 1A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (A): 200 μm.A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to transwell migration (B) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (B): 100 μm.(C) Effect of WDR63 knockdown on the migration of A549 cells was analyzed by single cell tracking with time-lapse microscopy at 10 min intervals for 4 h. Scale bar: 200 μm. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA.(D) A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to transwell invasion assay. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to transwell migration (E) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar : 100 μm.A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to single cell tracking (F) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar 200 μm.A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to transwell invasion (G) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar : 100 μm.(H) A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#2 (each also expressing luciferase) were injected via tail vein into nude mice (n=6 for each group). Three weeks after injection, tumor formation was monitored by bioluminescence imaging. Data shown are mean ± SD; *, P ˂ 0.05; One-way ANOVA.


Figure 2 ( A ) WDR63 mRNA and protein levels in A549 , H292 , and HCT116 cells transduced with empty vector ( PCDH ) or PCDH - p53 . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . ( B ) WDR63 mRNA and protein levels in A549 , H292 , and HCT116 cells transduced with control ( shctrl ) or p53 ( shp53 ) shRNA . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . ( C ) A549 cells expressing control or p53 shRNA were treated with doxorubicin ( Dox , 1μg / ml ) for the indicated periods of time . WDR63 mRNA and protein levels were then examined . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . ( D ) WDR63 mRNA levels in H1299 cells transfected with empty vector ( - ) , wild - type p53 , or the indicated p53 mutants . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . ( F ) Lysates from A549 cells were subjected to ChIP assay using control IgG or anti - p53 antibody . ChIP products were amplified by real - time PCR . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . ( G ) A549 cells were transfected with empty vector ( ctrl ) or Flag - p53 plus the indicated reporter constructs . Twenty - four hours later , reporter activity was measured . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . ( H ) A549 cells expressing control or p53 shRNA were transfected with pGL3 - BS1 and Renilla luciferase plasmids . Twenty - four hours later , cells were treated with or without Nutlin ( 10 μM ) for another 12 h . Reporter activity was then measured . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; two - tailed Student ' s t - test .

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

Figure 2(A) WDR63 mRNA and protein levels in A549, H292, and HCT116 cells transduced with empty vector (PCDH) or PCDH-p53. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; ***, P ˂ 0.001; two-tailed Student's t-test.(B) WDR63 mRNA and protein levels in A549, H292, and HCT116 cells transduced with control (shctrl) or p53 (shp53) shRNA. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.(C) A549 cells expressing control or p53 shRNA were treated with doxorubicin (Dox, 1μg/ml) for the indicated periods of time. WDR63 mRNA and protein levels were then examined. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.(D) WDR63 mRNA levels in H1299 cells transfected with empty vector (-), wild-type p53, or the indicated p53 mutants. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.(F) Lysates from A549 cells were subjected to ChIP assay using control IgG or anti-p53 antibody. ChIP products were amplified by real-time PCR. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.(G) A549 cells were transfected with empty vector (ctrl) or Flag-p53 plus the indicated reporter constructs. Twenty-four hours later, reporter activity was measured. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; ***, P ˂ 0.001; two-tailed Student's t-test.(H) A549 cells expressing control or p53 shRNA were transfected with pGL3-BS1 and Renilla luciferase plasmids. Twenty-four hours later, cells were treated with or without Nutlin (10 μM) for another 12 h. Reporter activity was then measured. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; two-tailed Student's t-test.


Figure 3 ( A ) A ~ 44 kD protein band specifically pulled down by GST - WDR63 from A549 cell lysates was identified as Arp2 by mass spectrometry analysis . The Arp2 peptide sequences obtained by MS are shown . ( B ) Lysates from HEK293T cells expressing HA - Arp2 alone or together with Flag - WDR63 were immunoprecipitated by anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments . ( C ) Lysates from HEK293T cells expressing GFP - WDR63 alone or together with Flag - Arp2 were immunoprecipitated by anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments . ( D ) Lysates from A549 cells were immunoprecipitated with control IgG or anti - WDR63 antibody , followed by western blot analysis . Data shown represent three independent experiments . ( E ) Purified GST or GST - Arp2 proteins immobilized on glutathione beads were incubated with purified Flag - WDR63 . Input and bead - bound proteins were analyzed by western blot . Data shown represent three independent experiments . ( F ) Purified GST or GST - WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2 . Input and bead - bound proteins were analyzed by western blot . Data shown represent three independent experiments . ( G ) Lysates from HEK293T cells expressing GFP - WDR63 alone or together with the indicated Flag - tagged each single subunit of the Arp2 / 3 complex were immunoprecipitated with anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments . ( H ) Purified Flag - tagged full - length or the indicated truncated WDR63 proteins immobilized on anti - Flag M2 beads were incubated with the purified Arp2 / 3 complex ( Cytoskeleton , Inc . ) in vitro . Input and beads - bound proteins were analyzed by western blot . Data shown represent three independent experiments .

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

Figure 3(A) A ~44 kD protein band specifically pulled down by GST-WDR63 from A549 cell lysates was identified as Arp2 by mass spectrometry analysis. The Arp2 peptide sequences obtained by MS are shown.(B) Lysates from HEK293T cells expressing HA-Arp2 alone or together with Flag-WDR63 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.(C) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with Flag-Arp2 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.(D) Lysates from A549 cells were immunoprecipitated with control IgG or anti-WDR63 antibody, followed by western blot analysis. Data shown represent three independent experiments.(E) Purified GST or GST-Arp2 proteins immobilized on glutathione beads were incubated with purified Flag-WDR63. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.(F) Purified GST or GST-WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.(G) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with the indicated Flag-tagged each single subunit of the Arp2/3 complex were immunoprecipitated with anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.(H) Purified Flag-tagged full-length or the indicated truncated WDR63 proteins immobilized on anti-Flag M2 beads were incubated with the purified Arp2/3 complex (Cytoskeleton, Inc.) in vitro. Input and beads-bound proteins were analyzed by western blot. Data shown represent three independent experiments.


Figure 4 ( A ) Immunolocalization of Arp2 in a wound - healing assay . A549 cells expressing control or WDR63 were serum - starved for 6 h before they were scratched . Cells were then treated with or without 10 % FBS for 30 min , followed by immunofluorescence assay . The branched actin networks were shown by immunostaining with anti - Cortactin antibody . Data shown represent three independent experiments . Scale bar : 20 μm . The lamellipodia formation was quantified by measuring the length of the outer margin of lamellipodia in individual cell ( n = 20 for each condition ) , and expressed as the proportion of the total length of the cell perimeter . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; One - way ANOVA . ( B and C ) Effects of full - length WDR63 ( B ) and WDR63 ( 1 - 200 ) ( C ) on Arp2 / 3 - mediated actin polymerization were examined by pyrene actin polymerization assays . Various concentrations of WDR63 ( B ) or WDR63 ( 1 - 200 ) ( C ) were incubated with 2 μM actin ( 50 % pyrene - labeled ) , 10 nM Arp2 / 3 and 400 nM GST - VCA as indicated . Data shown represent three independent experiments . ( D ) Effect of WDR63 on Arp2 / 3 - mediated branched actin polymerization was analyzed by electron microscopy . 0 . 6 μM WDR63 was incubated with 2 μM actin , 10 nM Arp2 / 3 and 400 nM GST - VCA as indicated . Actin structures were then stabilized with addition of 2μM phalloidin , followed by electron microscopy analysis . The arrows indicate actin branch junctions . The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments . For each condition , over 150 actin filaments were counted . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . Scale bar : 200 nm . ( E and F ) Increasing amounts of purified Flag - WDR63 ( 0 . 1 μM , 0 . 2 μM , and 0 . 4 μM ) ( E ) or Flag - WDR63 ( 1 - 200 ) ( 0 . 25 μM , 0 . 5 μM , and 1 μM ) ( F ) was incubated with the Arp2 / 3 complex ( 10 nM ) and GST - VCA as indicated in vitro , followed by GST pull - down assay . Data shown represent three independent experiments .

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

Figure 4(A) Immunolocalization of Arp2 in a wound-healing assay. A549 cells expressing control or WDR63 were serum-starved for 6 h before they were scratched. Cells were then treated with or without 10% FBS for 30 min, followed by immunofluorescence assay. The branched actin networks were shown by immunostaining with anti-Cortactin antibody. Data shown represent three independent experiments. Scale bar: 20 μm. The lamellipodia formation was quantified by measuring the length of the outer margin of lamellipodia in individual cell (n=20 for each condition), and expressed as the proportion of the total length of the cell perimeter. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; One-way ANOVA.(B and C) Effects of full-length WDR63 (B) and WDR63 (1-200) (C) on Arp2/3-mediated actin polymerization were examined by pyrene actin polymerization assays. Various concentrations of WDR63 (B) or WDR63 (1-200) (C) were incubated with 2 μM actin (50% pyrene-labeled), 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Data shown represent three independent experiments.(D) Effect of WDR63 on Arp2/3-mediated branched actin polymerization was analyzed by electron microscopy. 0.6 μM WDR63 was incubated with 2 μM actin, 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Actin structures were then stabilized with addition of 2μM phalloidin, followed by electron microscopy analysis. The arrows indicate actin branch junctions. The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments. For each condition, over 150 actin filaments were counted. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test. Scale bar: 200 nm.(E and F) Increasing amounts of purified Flag-WDR63 (0.1 μM, 0.2 μM, and 0.4 μM) (E) or Flag-WDR63 (1-200) (0.25 μM, 0.5 μM, and 1 μM) (F) was incubated with the Arp2/3 complex (10 nM) and GST-VCA as indicated in vitro, followed by GST pull-down assay. Data shown represent three independent experiments.


Figure 5A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bar in ( A ) : 200 μm . A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA Lysates from these cells were also analyzed by western blot ( B ) . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significanceA549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to transwell migration ( C ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bars in ( C : 100 μm . A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to transwell invasion ( D ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bars 100 μm . A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell migration ( E ) The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm . A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Lysates from these cells were also analyzed by western blot ( F ) . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell invasion ( G ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

Figure 5A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bar in (A): 200 μm.A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA Lysates from these cells were also analyzed by western blot (B). Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significanceA549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell migration (C) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars in (C : 100 μm.A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars 100 μm.A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Lysates from these cells were also analyzed by western blot (F). Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA.A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (G) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


Figure 6A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( A ) : 200 μm . A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 Lysates from these cells were also analyzed by western blot ( B ) . A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell migration ( C ) The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bars 100 μm . A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell invasion ( D ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bars 100 μm . A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell migration ( E ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm . A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell invasion ( F ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm . ( G ) A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 ( each also expressing luciferase ) were injected via tail vein into nude mice ( n = 6 for each group ) . Three weeks after injection , tumor formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

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

Figure 6A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (A): 200 μm.A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 Lysates from these cells were also analyzed by western blot (B).A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell migration (C) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm.A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm.A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (F) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.(G) A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 (each also expressing luciferase) were injected via tail vein into nude mice (n=6 for each group). Three weeks after injection, tumor formation was monitored by bioluminescence imaging. Data shown are mean ± SD; *, P ˂ 0.05; One-way ANOVA.


Figure 1Immunoblot ( IB ) analyses of the levels of β - catenin and RAS in DLD1 , WT - HCT116 , MT - HCT116 , HepG2 , and SW48 cells transfected with Myc - axin for 36 hrEffects of β - catenin knock - down on polyubiquitination - dependent degradation of RAS . IB analyses of MT - HCT116 cells transfected with control or two independent β - catenin siRNAs for 48 hrPolyubiquitination assays of MT - HCT116 cells cotransfected with two independent β - catenin siRNAs and HA - ubiquitin ( Ub ) for 72 hr in the presence of the proteasomal inhibitor MG132 , and WCLs were immunoprecipitated with an anti - pan - RAS antibodyIB analyses of HEK293 cells transfected with WT - or S33Y - Flag - β - catenin with or without HA - GSK3β for 36 hrIB analyses of HEK293 cells transfected with β - catenin siRNA or control siRNA for 48 h and then treated with L - cell - conditioned medium ( L - CM ) or Wnt3a - CM for 8 hrEffects of KYA1797K or XAV939 on degradation of β - catenin and RAS in isogenic HCT116 cells harboring WT - or MT - CTNNB1 . IB analyses of β - catenin and RAS levels in WT - or MT - HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hrICC analyses of WT - or MT - HCT116 cells treated with 20 µM of KYA1797K for 24 hr . Scale bars , 10 µmFor polyubiquitination assays , WT - or MT - HCT116 cells were transfected with FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132 . WCLs were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB analyses to detect each proteinThe 3 - ( 4 , 5 - dimethylthiazol - 2yl ) - 2 - 5 - diphenyltetrazolium bromide ( MTT ) assays were performed for measurements of growth of WT - or MT - HCT116 cells treated with the indicated concentrations of KYA1797K . Data are presented as the mean ± SD ( n = 3 )

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Figure 1Immunoblot (IB) analyses of the levels of β-catenin and RAS in DLD1, WT-HCT116, MT-HCT116, HepG2, and SW48 cells transfected with Myc-axin for 36 hrEffects of β-catenin knock-down on polyubiquitination-dependent degradation of RAS. IB analyses of MT-HCT116 cells transfected with control or two independent β-catenin siRNAs for 48 hrPolyubiquitination assays of MT-HCT116 cells cotransfected with two independent β-catenin siRNAs and HA-ubiquitin (Ub) for 72 hr in the presence of the proteasomal inhibitor MG132, and WCLs were immunoprecipitated with an anti-pan-RAS antibodyIB analyses of HEK293 cells transfected with WT- or S33Y-Flag-β-catenin with or without HA-GSK3β for 36 hrIB analyses of HEK293 cells transfected with β-catenin siRNA or control siRNA for 48 h and then treated with L-cell-conditioned medium (L-CM) or Wnt3a-CM for 8 hrEffects of KYA1797K or XAV939 on degradation of β-catenin and RAS in isogenic HCT116 cells harboring WT- or MT-CTNNB1. IB analyses of β-catenin and RAS levels in WT- or MT-HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hrICC analyses of WT- or MT-HCT116 cells treated with 20 µM of KYA1797K for 24 hr. Scale bars, 10 µmFor polyubiquitination assays, WT- or MT-HCT116 cells were transfected with FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132. WCLs were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses to detect each proteinThe 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assays were performed for measurements of growth of WT- or MT-HCT116 cells treated with the indicated concentrations of KYA1797K. Data are presented as the mean ± SD (n = 3)


Figure 2Analyses of the interaction between β - catenin and RAS . WCLs of DLD1 cells were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB analysesIB analyses of indicated proteins in the samples immunoprecipitated with anti - β - catenin antibody with WCLs of HEK293 cells treated with Wnt3a - CMLeft panel : MT - HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses , respectively . Right panel : The cytosolic and nuclear fractions were immunoprecipitated with anti - β - catenin antibody and subjected to IB analysesβ - Catenin - RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L - CM or Wnt3a - CM . Scale bars , 20 µm . The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100 cellsIn vitro pulldown analyses to identify β - catenin binding region of RAS . Upper panel : Schematic of the HRAS full - length ( FL ) or two deletion mutants . Lower panel : Purified GST - HRAS - FL , - ΔHVR , or - Δ131 - 165 proteins were pulled - down with purified His - β - catenin - FL protein and subjected to IB analysesIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - FL , - ΔHVR , or - Δ131 - 165 for 32 hrIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - WT , - 2A , or - 2E for 32 hrUpper panel : Schematic of the β - catenin - FL or two deletion mutants . Lower panel : Purified His - β - catenin - FL , ΔN86 , or ΔC110 proteins were pull - down with purified GST - HRAS - FL protein and subjected to IB analysesIB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc - HRAS and GFP - β - catenin - FL , - ΔN86 , or - ΔC110 for 32

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Figure 2Analyses of the interaction between β-catenin and RAS. WCLs of DLD1 cells were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analysesIB analyses of indicated proteins in the samples immunoprecipitated with anti-β-catenin antibody with WCLs of HEK293 cells treated with Wnt3a-CMLeft panel: MT-HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses, respectively. Right panel: The cytosolic and nuclear fractions were immunoprecipitated with anti-β-catenin antibody and subjected to IB analysesβ-Catenin-RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L-CM or Wnt3a-CM. Scale bars, 20 µm. The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100 cellsIn vitro pulldown analyses to identify β-catenin binding region of RAS. Upper panel: Schematic of the HRAS full-length (FL) or two deletion mutants. Lower panel: Purified GST-HRAS-FL, -ΔHVR, or -Δ131-165 proteins were pulled-down with purified His-β-catenin-FL protein and subjected to IB analysesIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-FL, -ΔHVR, or -Δ131-165 for 32 hrIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-WT, -2A, or -2E for 32 hrUpper panel: Schematic of the β-catenin-FL or two deletion mutants. Lower panel: Purified His-β-catenin-FL, ΔN86, or ΔC110 proteins were pull-down with purified GST-HRAS-FL protein and subjected to IB analysesIB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc-HRAS and GFP-β-catenin-FL, -ΔN86, or -ΔC110 for 32 hr


Figure 3Fluorescence spectroscopy analyses of the interaction between β - catenin and KRAS using purified recombinant proteins , intact β - catenin and KRAS . Molar ratios of β - catenin and KRAS are presented . A . U . , absorbance unitsNuclear magnetic resonance ( NMR ) titration for 15N - labeled KRAS ( full - length , 1 - 188 ) with β - catenin ( C - terminal fragment , 661 - 781 ) . The titration points are indicated in red ( 1 : 0 ) , green ( 1 : 5 ) , and blue ( 1 : 10 ) . A gradual perturbation in chemical shift is indicated by the

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Figure 3Fluorescence spectroscopy analyses of the interaction between β-catenin and KRAS using purified recombinant proteins, intact β-catenin and KRAS. Molar ratios of β-catenin and KRAS are presented. A.U., absorbance unitsNuclear magnetic resonance (NMR) titration for 15N-labeled KRAS (full-length, 1-188) with β-catenin (C-terminal fragment, 661-781). The titration points are indicated in red (1:0), green (1:5), and blue (1:10). A gradual perturbation in chemical shift is indicated by the arrow


Figure 4Effects of KYA1797K on the interaction and phosphorylation of β - catenin and pan - RAS . WT - or MT - HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132 . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB analysesEffects of Wnt3a - CM treatment on the formation of β - catenin destruction complex and phosphorylation of β - catenin and RAS in HEK293 cells treated with L - CM or Wnt3a - CM in the presence of MG132 for 8 hr . WCLs were immunoprecipitated with β - catenin or pan - RAS antibodies and subjected to IB analysesIB analyses were performed to detect β - catenin and pan - RAS in DLD1 cells cotransfected with FLAG - β - catenin - WT or - MT and Myc - KRAS - WT or - 2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA - GSK3β for 24 hr ,

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Figure 4Effects of KYA1797K on the interaction and phosphorylation of β-catenin and pan-RAS. WT- or MT-HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analysesEffects of Wnt3a-CM treatment on the formation of β-catenin destruction complex and phosphorylation of β-catenin and RAS in HEK293 cells treated with L-CM or Wnt3a-CM in the presence of MG132 for 8 hr. WCLs were immunoprecipitated with β-catenin or pan-RAS antibodies and subjected to IB analysesIB analyses were performed to detect β-catenin and pan-RAS in DLD1 cells cotransfected with FLAG-β-catenin-WT or -MT and Myc-KRAS-WT or -2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA-GSK3β for 24 hr, respectively


Figure 5IB analyses to determine the interaction between β - catenin and BBR peptide by in vitro pull - down assays using purified His - β - catenin and GST - BBR proteinsIB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG - β - catenin and GFP - BBR or - Mock in the presence of proteasome inhibitor , ALLN , for 24 hrEffects of BBR overexpression on KYA1797K - induced RAS degradation . I analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrEffects of BBR overexpression on KYA1797K - induced RAS degradation ICC ( D ; Scale bars , 10 µm ) analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT - HCT116 cells were cotransfected with GFP - Mock or - BBR and FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr , and WCLs were immunoprecipitated with pan - RAS antibodyIB analyses of IP with β - catenin antibody in MT - HCT116 cells transfected with GFP - BBR - WT , - 2E , or GFP - Mock in the presence of ALLN for 24 hrIB analyses of MT - HCT116 cells transfected with GFP - Mock , - BBR - WT , or - BBR - 2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT - HCT116 cells were transfected with GFP - Mock , - BBR - WT , or - BBR - 2E together with FLAG - Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB

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

Figure 5IB analyses to determine the interaction between β-catenin and BBR peptide by in vitro pull-down assays using purified His-β-catenin and GST-BBR proteinsIB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG-β-catenin and GFP-BBR or -Mock in the presence of proteasome inhibitor, ALLN, for 24 hrEffects of BBR overexpression on KYA1797K-induced RAS degradation. I analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrEffects of BBR overexpression on KYA1797K-induced RAS degradation ICC (D; Scale bars, 10 µm) analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT-HCT116 cells were cotransfected with GFP-Mock or -BBR and FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr, and WCLs were immunoprecipitated with pan-RAS antibodyIB analyses of IP with β-catenin antibody in MT-HCT116 cells transfected with GFP-BBR-WT, -2E, or GFP-Mock in the presence of ALLN for 24 hrIB analyses of MT-HCT116 cells transfected with GFP-Mock, -BBR-WT, or -BBR-2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT-HCT116 cells were transfected with GFP-Mock, -BBR-WT, or -BBR-2E together with FLAG-Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses


Figure 6Effects of KYA1797K and PTD - BBR on RAS degradation IB assay of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr , respectivelyEffects of KYA1797K and PTD - BBR o cell growth MTT assays of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr , respectively . Data are presented as the mean ± SD ( n = 3 ) Effects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growthEffects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformationEffects of cotreatments with KYA1797K and PTD - BBR on xenografted tumor growth . MT - HCT116 cells were subcutaneously injected in nude mice with subsequent i . p . injection of vehicle , 20 mg / kg of KYA1797K , or a cotreatment of PTD - BBR ( 25 mg / kg ) and KYA1797K ( 20 mg / kg ) for 20 d . Tumor volumes ( upper panel ) were measured every 4 days , and tumor weights ( lower panel ) were measured at the times of sacrifice . Data are presented as the mean ± SD ( four mice per group ) WCLs prepared from tumor tissues were immunoprecipitated with anti - β - catenin antibody . WCLs or co - IP samples were subjected to IB analysesIHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI . Scale bar , 20

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Figure 6Effects of KYA1797K and PTD-BBR on RAS degradation IB assay of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectivelyEffects of KYA1797K and PTD-BBR o cell growth MTT assays of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively. Data are presented as the mean ± SD (n = 3)Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growthEffects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformationEffects of cotreatments with KYA1797K and PTD-BBR on xenografted tumor growth. MT-HCT116 cells were subcutaneously injected in nude mice with subsequent i.p. injection of vehicle, 20 mg/kg of KYA1797K, or a cotreatment of PTD-BBR (25 mg/kg) and KYA1797K (20 mg/kg) for 20 d. Tumor volumes (upper panel) were measured every 4 days, and tumor weights (lower panel) were measured at the times of sacrifice. Data are presented as the mean ± SD (four mice per group)WCLs prepared from tumor tissues were immunoprecipitated with anti-β-catenin antibody. WCLs or co-IP samples were subjected to IB analysesIHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI. Scale bar, 20 µm