A ( left panel ) brightfield ( right panel ) of 5 - ALA DHA treated human cerebral organoids ( glioblastoma - like ) . Organoids were monitored on day 1 , day 5 , day 8 day 11 . One experiment is shown , representative of two independent experiments . Scale bar 500μm . B , C Image of GFP - positive areas of treated organoids . Four organoids per group were analyzed on day ( B ) d5 ( C ) d11 , normalized to day 1 . Data is shown as box plots ( 25th - 75th percentiles , median ) n = 4 .

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A Fluorescence (left panel) and brightfield (right panel) images of 5-ALA and DHA treated human cerebral tumor organoids (glioblastoma-like). Organoids were monitored on day 1, day 5, day 8 and day 11. One experiment is shown, representative of two independent experiments. Scale bar 500μm. B, C Image analysis and quantification of GFP-positive tumor areas of the treated organoids. Four organoids per group were analyzed on day (B) d5 and (C) d11, normalized to day 1. Data is shown as box plots (25th-75th percentiles, median) n=4. D


A Representative brightfield of 5 - ALA DHA treated - derived glioblastoma spheroids ( VBT92 ) . were taken on day 3 of culture left untreated or exposed to indicated treatments . Scale bar 500μm .

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A Representative brightfield images of 5-ALA and DHA treated patient-derived glioblastoma spheroids (VBT92). Images were taken on day 3 of culture left untreated or exposed to the indicated treatments. Scale bar 500μm.


B Re - plating of spheroids after 3 days of 5 - ALA DHA treatment ( duplicates ) . Representative brightfield are shown for glioblastoma VBT92 .

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B Re-plating of spheroids after 3 days of 5-ALA and DHA treatment (duplicates). Representative brightfield images are shown for glioblastoma VBT92.


C of re - adhesion capacity of re - plated VBT92 spheroids as assessed by crystal violet absorbance measurement . Data is shown as mean ± SD . Experiments were performed in quadruplicates .

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C Quantification of the re-adhesion capacity of re-plated VBT92 spheroids as assessed by crystal violet staining and absorbance measurement. Data is shown as mean ± SD. Experiments were performed in quadruplicates.


D Lipid peroxidation in VBT92 treated with 5 - ALA DHA . Lipid peroxidation was measured using BODIPY ™ 581 / 591 C11 green ( from red ) shift was determined using flow cytometry . Experiments were performed in triplicate ; values are shown as mean ± SD .

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D Lipid peroxidation in VBT92 cells treated with 5-ALA and DHA. Lipid peroxidation was measured using BODIPY™ 581/591 C11 staining and green (from red) fluorescence shift was determined using flow cytometry. Experiments were performed in triplicate; values are shown as mean ± SD.


B In vivo efficacy comparison of solvents ( control ) , 5 - ALA , artesunate ( ARS ) combined treatment in an orthotopic glioblastoma model . 11 - 14 days after stereotactic intracerebral injection of 2 x 105 VBT529 , mice received following treatments : Control ( solvents ) , 5 - ALA ( 80 mg / kg ) , ARS ( 40 mg / kg ) , or 5 - ALA plus ARS 5 times per week via intraperitoneal injection ( n ≥ 5 ) .

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B In vivo efficacy comparison of solvents (control), 5-ALA, artesunate (ARS) and combined treatment in an orthotopic glioblastoma model. 11-14 days after stereotactic intracerebral injection of 2 x 105 VBT529 cells, mice received the following treatments: Control (solvents), 5-ALA (80 mg/kg), ARS (40 mg/kg), or 5-ALA plus ARS 5 times per week via intraperitoneal injection (n ≥ 5).


C , D representative of VBT529 flank luminescence signal . 6 - 7 days after injection of 1 . 5 x 105 VBT529 into flank , mice received following treatments : Control ( solvents ) or 5 - ALA plus ARS 4 - 5 times per week for 5 via intraperitoneal injection ( n = 6 ) . After 4 days of treatment , concentrations of 5 - ALA ( 100 mg / kg ) ARS ( 50 mg / kg ) were increased to 120 mg / kg 60 mg / kg , respectively . 20 % dosage increase of both drugs was used as - , behaviour overall health of mice were not affected by both , lower as well as higher dosages . E , F representative of VBT529 brain luminescence signals . 6 - 7 days after stereotactic intracerebral injection of 1 . 5 x 105 VBT529 , mice received treatments as described in ( C D ) ( n = 6 ) .

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C, D Quantification and representative images of the VBT529 flank tumor luminescence signal. 6-7 days after injection of 1.5 x 105 VBT529 cells into the flank, mice received the following treatments: Control (solvents) or 5-ALA plus ARS 4-5 times per week for 5 weeks via intraperitoneal injection (n = 6). After 4 days of treatment, concentrations of 5-ALA (100 mg/kg) and ARS (50 mg/kg) were increased to 120 mg/kg and 60 mg/kg, respectively. The 20% dosage increase of both drugs was used as the body-weight, behaviour and overall health of the mice were not affected by both, the lower as well as higher dosages. E, F Quantification and representative images of VBT529 brain tumor luminescence signals. 6-7 days after stereotactic intracerebral injection of 1.5 x 105 VBT529 cells, mice received the treatments as described in (C and D) (n = 6).


G Kaplan - Meier survival curves of mice injected with VBT529 glioblastoma into flank brain ( same mice as in C - F ; n = 6 ) . Mice were treated with solvents ( control ) or 5 - ALA plus ARS ( same mice as in C - F ) .

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G Kaplan-Meier survival curves of mice injected with VBT529 glioblastoma cells into the flank and brain (same mice as in C-F; n = 6). Mice were treated with solvents (control) or 5-ALA plus ARS (same mice as in C-F).


A Mass spectrometric of LSD1 methylation . Immunoprecipitation ( IP ) was performed using anti - Flag antibody in HEK - 293T overexpressing Flag - tagged LSD1 , followed by liquid chromatography coupled tandem mass spectrometry ( LC - MS / MS ) . fragmentation of LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838 .

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A 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 CARM1 were detected by co - immunoprecipitation ( Co - IP ) with anti - Flag in HEK - 293T transfected with Flag - LSD1 or Flag - CARM1 , respectively .

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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 using anti - CARM1 antibody in MDA - MB - 231 .

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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 Flag - LSD1 purified from HEK - 293T . amounts of GST GST - tagged CARM1 were visualized by Coomassie Blue ( asterisk : GST - CARM1 ) .

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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 of GST - tagged LSD1 WT or four methylated arginine mutants ( R - A ) , or four methylated arginine mutants in combination ( 4RA ) , were incubated with GST - CARM1 in presence of 3H - SAM . Methylated were detected via autoradiography total amounts of were visualized by Coomassie Blue ( arrows : position of LSD1 ; arrowheads : position of CARM1 ) .

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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 . methylation of LSD1 was analyzed by immunoblotting with anti - LSD1 R838me2a antibody .

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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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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.


A Methylation of LSD1 was assessed in MCF10A , MCF7 MDA - MB - 231 by immunoblotting using anti - LSD1 R838me2a .

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A Methylation of LSD1 was assessed in MCF10A, MCF7 and MDA-MB-231 cells by immunoblotting using anti-LSD1 R838me2a.


expression of LSD1 in MDA - MB - 231 transfected with CARM1 shRNAs or control vector , were assessed by western blotting ( B ) ( Error bars represent mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - ) .

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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 of LSD1 in MDA - MB - 231 transfected with CARM1 shRNAs or control vector , were assessed by qPCR ( C ) ( Error bars represent mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - ) .

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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 were treated with indicated amounts of MS049 for 48 h , LSD1 R838me2a LSD1 were assessed by western blotting using indicated antibodies .

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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 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 .

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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 were transfected with control or CARM1 shRNAs then treated with MG132 or DMSO for 10 h , lysates were analyzed by western blotting .

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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 were transfected with CARM1 shRNA # 2 or control vector , then treated with CHX ( 100 μg / ml ) for indicated time ; LSD1 level was examined by western blotting .

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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 were transfected with control or CARM1 shRNAs then treated with MG132 for 10 h . lysates were immunoprecipitated by anti - LSD1 antibody then analysed by immunoblotting .

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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 were treated with increasing amounts of MS049 for 48 h MG132 for 10 h . ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti - LSD1 antibody .

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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 indicated time . LSD1 were examined by western blotting .

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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 were transfected with indicated plasmids then treated with MG132 for 10 h . lysates were immunoprecipitated using anti - Flag antibody then subjected to immunoblotting .

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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 expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h MG132 for 10 h . Co - IP was performed using anti - Flag then subjected to western blotting .

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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.


A Immunoprecipitation was performed using anti - Flag antibody in MM - 231 - shLSD1 - WT / R838A . interaction of LSD1 DUBs were detected by immunoblotting with indicated antibodies .

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A 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 were treated with indicated amounts of P5091 for 48 h , LSD1 were assessed by immunoblotting .

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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 were treated with P5091 ( 10 μM ) for 48 h MG132 for 10 h . LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti - LSD1 antibody .

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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 were transfected with USP7 WT or USP7 C223S , then treated with MG132 for 10 h . lysates were immunoprecipitated using anti - LSD1 antibody followed by immunoblotting .

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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 overexpressing Flag - tagged LSD1 WT / R838A after knockdown of endogenous LSD1 then treated with or without P5091 for 48 h MG132 for 10 h . lysates were immunoprecipitated by anti - Flag antibody then analysed by immunoblotting .

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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 were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h . lysates were immunoprecipitated with anti - LSD1 then subjected to immunoblotting .

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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 were treated with or without MS049 for 48 h MG132 for 10 h , lysates were immunoprecipitated using anti - LSD1 antibody then analysed by immunoblotting .

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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 were transfected with USP7 WT or USP7 C223S , then treated with or without MS049 for 48 h MG132 for 10 h . lysates were immunoprecipitated with anti - LSD1 antibody followed by immunoblotting .

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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.


Migration assays ( 24 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F ( A ) with or without MS049 treatment . Representative of migrated are shown . Data represent number of derived from mean of five fields ( Error bars indicate mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - ) .

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Migration 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 ( B ) with or without MS049 treatment . Representative of invaded are shown . Data represent number of derived from mean 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 - ) .

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 ( C ) with or without MS049 treatment . Representative of migrated are shown . Data represent number of derived from mean of five fields ( Error bars indicate mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - ) .

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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 ( D ) with or without MS049 treatment . Representative of invaded are shown . Data represent number of derived from mean 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 - ) .

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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 ( 1 × 106 ) were injected into tail veins of 5 - week - old nude mice , 7 later , mice were sacrificed with euthanasia lungs were fixed in paraformaldehyde solutions . 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 - ) .

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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 eosin . Scale bars , 100 μm .

F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.


MDA - MB - 231 were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody immunoprecipitated 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 - ) .

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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 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 were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody immunoprecipitated 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 - ) .

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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 were treated with MS049 or P5091 , real - time PCR was performed to E - cadherin ( C ) mRNA ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - ) .

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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 were treated with MS049 or P5091 , real - time PCR was performed to vimentin ( D ) mRNA ( Data are are presented as mean ± SD , n = 3 experimental replicates , * * P < 0 . 01 , ns = not significant , Student ' s t - ) .

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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 were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , immunoprecipitated 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 - ) .

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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 were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 or anti - H3K4me2 or anti - H3K9me2 antibody , immunoprecipitated 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 - ) .

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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).


A Typical pictures of immunohistochemical of LSD1 R838me2a , LSD1 CARM1 in breast cancer samples . Scale bars , 100 μm .

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A Typical pictures of the immunohistochemical staining of LSD1 R838me2a, LSD1 and CARM1 in breast cancer samples. Scale bars, 100 μm.


of samples with high level of LSD1 R838me2a ( B ) expression in different TNM stages of breast cancer ( n = 70 breast samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 ) .

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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 of samples with high level of , LSD1 ( C ) CARM1 ( D ) expression in different TNM stages of breast cancer ( n = 70 breast samples ; B , P = 0 . 0095 ; C , P = 0 . 002 ; D , P = 0 . 0024 ; χ2 ) .

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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).


correlation of LSD1 R838me2a with LSD1 ( E ) was statistically significant among breast cancer specimens ( n = 70 breast samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation ) . Note that some samples have overlapping scores . score was evaluated according to Feng et al .

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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 correlation of , CARM1 with LSD1 R838me2a ( F ) CARM1 with LSD1 ( G ) was statistically significant among breast cancer specimens ( n = 70 breast samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation ) . Note that some samples have overlapping scores . score was evaluated according to Feng et al .

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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.


A . Volcano blot representation of differently expressed genes in FCRLS - CD11b - positive Grn - / - microglia in comparison to wt microglia isolated from 5 . 5 - month - old mice ( , n = 5 ) . 116 genes out of 529 genes analyzed , are significantly changed , from these 58 genes are up - 58 genes are down regulated . Eight genes with highest fold change are highlighted .

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A. 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 . of significantly affected genes ( p < 0 . 05 ) in FCRLS - CD11b - positive Grn - / - microglia in comparison to Grn + / + microglia isolated from 5 . 5 - month - old mice ( n = 5 per genotype ) . For significantly affected genes of Grn - / - microglia , mRNA expression data of Trem2 - / - microglia in comparison to corresponding wt microglia were taken from previously published data ( Mazaheri et al , 2017 ) . for each gene sample were normalized to 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 ) .

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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 of FCRLS - CD11b - positive Grn - / - microglia normalized to wt in comparison to published normalized data set of Trem2 - / - microglia ( Mazaheri et al , 2017 ) .

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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).


A . Immunoblot of ApoE , CLEC7A , TREM2 CD68 in lysates of acutely isolated microglia from Grn - / - Grn + / + mice ( 9 months of , n = 3 per genotype , ) . IBA1 was used as loading control .

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A. 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 . of expression normalized to of Grn + / + microglia ( n = 3 ) . Data represent mean ± SD .

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B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n=3). Data represent the mean ± SD.


C . ELISA mediated of sTREM2 in brain homogenates of Grn - / - Grn + / + mice Data are shown as mean ± ( n = 3 - 6 ) . D . ELISA mediated of sTREM2 in serum of Grn - / - Grn + / + mice . Data are shown as mean ± ( n = 4 - 13 ) .

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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 - / - Trem2 - / - mice . ( 9 months of , ) F . of P2RY12 positive microglia . Data are shown as mean ± SD ( n = 3 per genotype , , except one for Trem2 - / - ) .

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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 of secreted PGRN ApoE ( ApoEmed ) in conditioned media PGRN , ApoE , CLEC7A , P2RY12 TREM2 in lysates of BV2 wild type ( Grnwt ) PGRN deficient ( Grnmut ) . Soluble amyloid precursor ( APPs ) calnexin were used as a loading control .

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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 . of Immunoblots normalized to BV2 Grnwt ( n = 3 - 5 ) . Data represent mean ± SD .

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H. Quantification of Immunoblots normalized to BV2 Grnwt levels (n=3-5). Data represent the mean ± SD.


I . ELISA mediated of sTREM2 in conditioned media of BV2 Grnwt Grnmut . Data represent mean ±

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I. ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SD


J . of relative mRNA of Apoe , Clec7a , P2ry12 Trem2 in BV2 Grnwt Grnmut . Data represent mean ± SD .

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J. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD.


A . Flow cytometric of phagocytic capacity in BV2wt BV2mut using pHrodo green E . coli as target particles . Phagocytosis was terminated after 30 60 minutes ( min ) of incubation . Data are presented as mean of positive for pHrodo uptake ± S . D ( n = 3 ) .

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A. 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 in an ex vivo model . Immunofluorescence of cultured Grn + / + Grn - / - organotypic brain at 7 DIV immunostained using microglial marker CD68 . Nuclei were counterstained using DAPI . 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 reveals that Grn - / - microglia migrate larger distances than Grn + / + . Data are shown as mean ± SD ( n = 3 independent experiments ) displays an increased number of migrating Grn - / - compared to Grn + / + . Data are shown as mean ± SD ( n = 3 independent experiments )

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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 absence of PGRN . Left : IBA1 - stained microglia clustering around X - 34 - positive amyloid plaque cores in 4 - month - old APPPS1 / Grn + / + mice . Right : - matched APPPS1 / Grn - / - mice display microglial hyper - clustering around amyloid plaques compared to APPPS1 / Grn + / + . Dotted boxes indicate that is shown at higher magnification . Scale bars indicate 10µm 3µm in inset . Immunohistochemical of IBA1 positive microglial number per

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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


A . TSPO - µPET in rodents : Axial slices ( A1 , A2 , A3 ) of averaged % - TSPO - PET differences between Grn - / - or Trem2 - / - mice wt indicate increased microglial activity in cerebrum of Grn - / - mice ( hot color scale ) decreased microglial activity in 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 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 .

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A. 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 of subjected to TSPO - PET confirmed GRN mutation non - GRN mutation carrier . PGRN was measured by ELISA normalized to serum of a healthy control .

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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 : Visual interpretation of axial TSPO - PET SUVR in upper cranial layers reveals increased microglial activity in frontal parietal cortices ( arrows ) of a FTLD with a GRN loss of function mutation in comparison to a FTLD without a GRN mutation . PET results are illustrated upon an MRI template ; 60 - 80 min p . i . emission recording ; global mean scaling . Both were low - affinity binder .

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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.


FDG - µPET in rodents : Axial slices ( as indicated in Fig . 4A ) of averaged % - FDG - µPET differences between Grn - / - or Trem2 - / - mice wt indicate decreased glucose metabolism in cerebrum of Grn - / - 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 mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 6 at 11 months ; wt n = 10 at 8 - 11 months ) .

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FDG-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).


A549 expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to wound - healing ( A ) assays . shown 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 .

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A549 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 expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell migration ( B ) assays . shown 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 .

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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 migration of A549 was analyzed by single tracking with time - lapse microscopy at 10 min intervals for 4 h . Scale bar : 200 μm . migration speed , persistence , mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell invasion . shown 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 O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell migration ( E ) assays . shown are representative of three independent experiments . migration speed , persistence , mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik 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 .

O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to single tracking ( F ) assays . shown are representative of three independent experiments . migration speed , persistence , mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik 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 .

O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell invasion ( G ) assays . shown are representative of three independent experiments . migration speed , persistence , mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik 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 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 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 after injection , formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A ) WDR63 mRNA in A549 , H292 , HCT116 transduced with empty vector ( PCDH ) or PCDH - p53 . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; two - Student ' s t - .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 in A549 , H292 , HCT116 transduced with control ( shctrl ) or p53 ( shp53 ) shRNA . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - Student ' s t - .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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) 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 expressing control or p53 shRNA were treated with doxorubicin ( Dox , 1μg / ml ) for indicated periods of time . WDR63 mRNA were then examined . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - Student ' s t - .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 in H1299 transfected with empty vector ( - ) , wild - type p53 , or indicated p53 mutants . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - Student ' s t - .

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(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 were subjected to ChIP 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 - Student ' s t - .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 were transfected with empty vector ( ctrl ) or Flag - p53 plus 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 - Student ' s t - .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 expressing control or p53 shRNA were transfected with pGL3 - BS1 Renilla luciferase plasmids . Twenty - four hours later , 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 - Student ' s t - .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A ) A ~ 44 kD band specifically pulled down by GST - WDR63 from A549 lysates was identified as Arp2 by mass spectrometry . Arp2 peptide sequences obtained by MS are shown .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O

(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 expressing HA - Arp2 alone or together with Flag - WDR63 were immunoprecipitated by anti - Flag antibody , followed by western blot . Data shown represent three independent experiments .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(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 expressing GFP - WDR63 alone or together with Flag - Arp2 were immunoprecipitated by anti - Flag antibody , followed by western blot . 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 were immunoprecipitated with control IgG or anti - WDR63 antibody , followed by western blot . Data shown represent three independent experiments .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(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 immobilized on glutathione beads were incubated with purified Flag - WDR63 . Input bead - bound were analyzed by western blot . Data shown represent three independent experiments .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(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 immobilized on glutathione beads were incubated with purified Arp2 . Input bead - bound were analyzed by western blot . Data shown represent three independent experiments .

O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(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 expressing GFP - WDR63 alone or together with indicated Flag - tagged each single subunit of Arp2 / 3 were immunoprecipitated with anti - Flag antibody , followed by western blot . Data shown represent three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(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 - or indicated truncated WDR63 immobilized on anti - Flag M2 beads were incubated with purified Arp2 / 3 ( Cytoskeleton , Inc . ) in vitro . Input beads - bound were analyzed by western blot . Data shown represent three independent experiments .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( A ) Immunolocalization of Arp2 in a wound - healing . A549 expressing control or WDR63 were serum - starved for 6 h before they were scratched . were then treated with or without 10 % FBS for 30 min , followed by immunofluorescence . branched actin networks were shown by immunostaining with anti - Cortactin antibody . Data shown represent three independent experiments . Scale bar : 20 μm . lamellipodia formation was quantified by measuring of outer margin of lamellipodia in ( n = 20 for each condition ) , expressed as proportion of total of perimeter . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; One - way ANOVA .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 C ) Effects of full - WDR63 ( B ) 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 / 3 400 nM GST - VCA as indicated . Data shown represent three independent experiments .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(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 / 3 400 nM GST - VCA as indicated . Actin structures were then stabilized with addition of 2μM phalloidin , followed by electron microscopy . arrows indicate actin branch junctions . of branched actin filaments was expressed as of number of actin filaments with branch junctions to 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 - Student ' s t - . Scale bar : 200 nm .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 F ) Increasing amounts of purified Flag - WDR63 ( 0 . 1 μM , 0 . 2 μM , 0 . 4 μM ) ( E ) or Flag - WDR63 ( 1 - 200 ) ( 0 . 25 μM , 0 . 5 μM , 1 μM ) ( F ) was incubated with Arp2 / 3 ( 10 nM ) GST - VCA as indicated in vitro , followed by GST pull - down . Data shown represent three independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


A549 expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 Arp2 shRNA were subjected to wound - healing ( A ) assays . shown 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 .

O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A549 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 expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 Arp2 shRNA Lysates from these were also analyzed by western blot ( B ) . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no

O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 significance


A549 expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 Arp2 shRNA were subjected to transwell migration ( C ) assays . shown 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 .

O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A549 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 expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 Arp2 shRNA were subjected to transwell invasion ( D ) assays . shown 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 .

O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - or truncated WDR63 as indicated . were then subjected to transwell migration ( E ) shown 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 .

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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 expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - or truncated WDR63 as indicated . Lysates from these 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 .

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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 expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - or truncated WDR63 as indicated . were then subjected to transwell invasion ( G ) assays . shown 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 .

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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.


A549 expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to wound - healing ( A ) assays . shown 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 .

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A549 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 expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 Lysates from these were also analyzed by western blot ( B ) .

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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 expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell migration ( C ) shown 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 .

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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 expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell invasion ( D ) assays . shown 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 .

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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 expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - or truncated WDR63 as indicated . were then subjected to transwell migration ( E ) assays . shown 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 .

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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 expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - or truncated WDR63 as indicated . were then subjected to transwell invasion ( F ) assays . shown 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 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 after injection , formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

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(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.


Immunoblot ( IB ) analyses of of β - catenin RAS in DLD1 , WT - HCT116 , MT - HCT116 , HepG2 , SW48 transfected with Myc - axin for 36

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Immunoblot (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 hr


Effects of β - catenin knock - down on polyubiquitination - dependent degradation of RAS . IB analyses of MT - HCT116 transfected with control or two independent β - catenin siRNAs for 48

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Effects 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 hr


Polyubiquitination assays of MT - HCT116 cotransfected with two independent β - catenin siRNAs HA - ubiquitin ( Ub ) for 72 hr in presence of proteasomal inhibitor MG132 , WCLs were immunoprecipitated with an anti - pan - RAS

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Polyubiquitination 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 antibody


IB analyses of HEK293 transfected with WT - or S33Y - Flag - β - catenin with or without HA - GSK3β for 36

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IB analyses of HEK293 cells transfected with WT- or S33Y-Flag-β-catenin with or without HA-GSK3β for 36 hr


IB analyses of HEK293 transfected with β - catenin siRNA or control siRNA for 48 h then treated with L - - conditioned medium ( L - CM ) or Wnt3a - CM for 8

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IB 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 hr


Effects of KYA1797K or XAV939 on degradation of β - catenin RAS in isogenic HCT116 harboring WT - or MT - CTNNB1 . IB analyses of β - catenin RAS in WT - or MT - HCT116 treated with 5 or 20 µM of KYA1797K or XAV939 for 24

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Effects 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 hr


ICC analyses of WT - or MT - HCT116 treated with 20 µM of KYA1797K for 24 hr . Scale bars , 10

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ICC analyses of WT- or MT-HCT116 cells treated with 20 µM of KYA1797K for 24 hr. Scale bars, 10 µm


For polyubiquitination assays , WT - or MT - HCT116 were transfected with FLAG - Ub for 24 hr then treated with 20 µM of KYA1797K for 24 hr in presence of MG132 . WCLs were immunoprecipitated with β - catenin or pan - RAS antibody subjected to IB analyses to detect each

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For 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 protein


3 - ( 4 , 5 - dimethylthiazol - 2yl ) - 2 - 5 - diphenyltetrazolium bromide ( MTT ) assays were performed for measurements of growth of WT - or MT - HCT116 treated with indicated concentrations of KYA1797K . Data are presented as mean ± SD ( n = 3 )

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The 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)


Analyses of interaction between β - catenin RAS . WCLs of DLD1 were immunoprecipitated with β - catenin or pan - RAS antibody subjected to IB

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Analyses 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 analyses


IB analyses of indicated in samples immunoprecipitated with anti - β - catenin antibody with WCLs of HEK293 treated with Wnt3a -

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IB analyses of indicated proteins in the samples immunoprecipitated with anti-β-catenin antibody with WCLs of HEK293 cells treated with Wnt3a-CM


Left panel : MT - HCT116 extracts were fractionated as portions subjected to IB analyses , respectively . Right panel : fractions were immunoprecipitated with anti - β - catenin antibody subjected to IB

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Left 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 analyses


β - Catenin - RAS interaction was visualized by in situ proximity ligation in HEK293 treated with L - CM or Wnt3a - CM . Scale bars , 20 µm . proximity ligation signal was quantified in 10 randomly distributed microscopic that contained > 100

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β-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 cells


In vitro pulldown analyses to identify β - catenin binding region of RAS . Upper panel : Schematic of HRAS full - ( FL ) or two deletion mutants . Lower panel : Purified GST - HRAS - FL , - ΔHVR , or - Δ131 - 165 were pulled - down with purified His - β - catenin - FL subjected to IB

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In 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 analyses


IB analyses of IP with FLAG antibody in WCLs of HEK293 cotransfected with FLAG - β - catenin Myc - HRAS - FL , - ΔHVR , or - Δ131 - 165 for 32

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IB 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 hr


IB analyses of IP with FLAG antibody in WCLs of HEK293 cotransfected with FLAG - β - catenin Myc - HRAS - WT , - 2A , or - 2E for 32

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IB 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 hr


Upper panel : Schematic of β - catenin - FL or two deletion mutants . Lower panel : Purified His - β - catenin - FL , ΔN86 , or ΔC110 were pull - down with purified GST - HRAS - FL subjected to IB

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Upper 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 analyses


IB analyses of IP with Myc antibody with WCLs of HEK293 cotransfected with Myc - HRAS GFP - β - catenin - FL , - ΔN86 , or - ΔC110 for 32

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IB 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


spectroscopy analyses of interaction between β - catenin KRAS using purified recombinant , intact β - catenin KRAS . Molar ratios of β - catenin KRAS are presented . A . U . , absorbance

Fluorescence 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 units


magnetic resonance ( NMR ) titration for 15N - labeled KRAS ( full - , 1 - 188 ) with β - catenin ( C - terminal fragment , 661 - 781 ) . titration points are indicated in red ( 1 : 0 ) , green ( 1 : 5 ) , blue ( 1 : 10 ) . A gradual perturbation in chemical shift is indicated by

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Nuclear 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


Effects of KYA1797K on interaction phosphorylation of β - catenin pan - RAS . WT - or MT - HCT116 were treated with 20 µM of KYA1797K in presence of MG132 . WCLs were immunoprecipitated with pan - RAS antibody subjected to IB

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Effects 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 analyses


Effects of Wnt3a - CM treatment on formation of β - catenin destruction phosphorylation of β - catenin RAS in HEK293 treated with L - CM or Wnt3a - CM in presence of MG132 for 8 hr . WCLs were immunoprecipitated with β - catenin or pan - RAS antibodies subjected to IB

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Effects 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 analyses


IB analyses were performed to detect β - catenin pan - RAS in DLD1 cotransfected with FLAG - β - catenin - WT or - MT Myc - KRAS - WT or - 2A for 24 hr then treated or transfected with 20 µM of KYA1797K or HA - GSK3β for 24 hr ,

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IB 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


IB analyses to determine interaction between β - catenin BBR peptide by in vitro pull - down assays using purified His - β - catenin GST - BBR

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IB 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 proteins


IB analyses of IP with FLAG antibody WCLs from HEK293 cotransfected with FLAG - β - catenin GFP - BBR or - Mock in presence of proteasome inhibitor , ALLN , for 24

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IB 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 hr


Effects of BBR overexpression on KYA1797K - induced RAS degradation . I analyses to detect of RAS in MT - HCT116 transfected with GFP - Mock or - BBR for 24 hr then treated with 20 µM of KYA1797K for 24

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Effects 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 hr


Effects of BBR overexpression on KYA1797K - induced RAS degradation ICC ( D ; Scale bars , 10 µm ) analyses to detect of RAS in MT - HCT116 transfected with GFP - Mock or - BBR for 24 hr then treated with 20 µM of KYA1797K for 24

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Effects 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 hr


MT - HCT116 were cotransfected with GFP - Mock or - BBR FLAG - Ub for 24 hr then treated with 20 µM of KYA1797K in presence of MG132 for 8 hr , WCLs were immunoprecipitated with pan - RAS

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MT-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 antibody


IB analyses of IP with β - catenin antibody in MT - HCT116 transfected with GFP - BBR - WT , - 2E , or GFP - Mock in presence of ALLN for 24

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IB 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 hr


IB analyses of MT - HCT116 transfected with GFP - Mock , - BBR - WT , or - BBR - 2E for 24 hr then treated with 20 µM of KYA1797K for 24

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IB 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 hr


MT - HCT116 were transfected with GFP - Mock , - BBR - WT , or - BBR - 2E together with FLAG - Ub with or without 20 µM of KYA1797K for 24 hr MG132 was treated for 8 hr before harvesting . WCLs were immunoprecipitated with pan - RAS antibody subjected to IB

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O 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-MEASURED_VAR O O O O

MT-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


Effects of KYA1797K PTD - BBR on RAS degradation IB of MT - HCT116 or SW48 treated with indicated conditions for 24 96 hr ,

O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

Effects 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, respectively