E , F ( E ) ROS levels ( DHE staining , PE 582 / 15 nm - MFI ) and ( F ) cell survival of DHA ( 0 . 5μM ) , 5 - ALA ( 0 . 25mM ) or Ppox inhibitor ( 10μM ) treated human neuroblastoma cells ( SHSY5Y ) . Fluorescence of DHE ( ROS levels ) and cell numbers ( survival ) were assessed by flow cytometry and automated cell counting . The experiments were performed two times , in triplicate each .

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E, F (E) ROS levels (DHE staining, PE 582/15 nm - MFI) and (F) cell survival of DHA (0.5μM), 5-ALA (0.25mM) or Ppox inhibitor (10μM) treated human neuroblastoma cells (SHSY5Y). Fluorescence of DHE (ROS levels) and cell numbers (survival) were assessed by flow cytometry and automated cell counting. The experiments were performed two times, in triplicate each.


C , D ( C ) Fluorescence and ( D ) brightfield images of DMSO ( control ) , 5 - ALA ( 0 . 0625mM ) , DHA ( 1μM ) , or 5 - ALA and DHA ( 0 . 0625mM and 1μM ) treated tumor organoids ( CNS - PNET - like , c - MYC overexpression ) . Organoids were imaged on day 1 , day 5 and day 7 . One experiment is shown , representative of four independent experiments .

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C, D (C) Fluorescence and (D) brightfield images of DMSO (control), 5-ALA (0.0625mM), DHA (1μM), or 5-ALA and DHA (0.0625mM and 1μM) treated tumor organoids (CNS-PNET-like, c-MYC overexpression). Organoids were imaged on day 1, day 5 and day 7. One experiment is shown, representative of four independent experiments.


G ROS / DHE staining and flow cytometry analysis of dissociated tumor organoids ( PE , 582 / 15 nm ) . Mean fluorescence intensities ( MFI ) of DHE stained GFP + tumor cells and GFP - control cells are indicated . n = 2 , values are mean ± SD .

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G ROS/ DHE staining and flow cytometry analysis of dissociated tumor organoids (PE, 582/15 nm). Mean fluorescence intensities (MFI) of DHE stained GFP+ tumor cells and GFP- control cells are indicated. n=2, values are mean ± SD.


H Quantification of γH2AX + cells in cerebral tumor organoids . 6 cryo - sections of 3 organoids each were stained for γH2AX , scanned and 25 regions of interest ( ROI 2 . 500μm2 ) were analyzed per condition . Raw data were normalized to organoid area ( per ROI ) and are shown as box plots ( 25th - 75th percentiles , median ) .

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H Quantification of γH2AX+ cells in cerebral tumor organoids. 6 cryo-sections of 3 organoids each were stained for γH2AX, scanned and 25 regions of interest (ROI 2.500μm2) were analyzed per condition. Raw data were normalized to organoid area (per ROI) and are shown as box plots (25th-75th percentiles, median).


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 .

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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 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 .

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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 and DHA treatment ( duplicates ) . Representative brightfield images 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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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

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 ) .

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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 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 ) .

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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 . And 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 and MDA - MB - 231 cells by immunoblotting using anti - LSD1 R838me2a .

A 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the indicated time . LSD1 were examined by western blotting analysis .

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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 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 .

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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 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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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 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 .

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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 cells were treated with the indicated amounts of P5091 for 48 h , LSD1 protein levels were assessed by immunoblotting .

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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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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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 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 ) .

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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 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 ) .

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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 ) .

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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 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 ) .

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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 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 ) .

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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 and eosin staining . Scale bars , 100 μm .

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


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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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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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 the immunohistochemical staining of LSD1 R838me2a , LSD1 and 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.


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 ) .

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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 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 ) .

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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).


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 .

O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-DISEASE I-DISEASE O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

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 .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-DISEASE I-DISEASE O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

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 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 .

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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 . 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 ) .

O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

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 ) .

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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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O

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

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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 - / - 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 - / - ) .

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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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O

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 .

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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 ±

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

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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 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 )

O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD O O O B-CELL_TYPE B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

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 O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O 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-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O

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 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 .

O O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O B-CELL_TYPE B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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. 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 .

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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 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 O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM B-DISEASE B-ORGANISM O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O B-CELL_TYPE B-EXP_ASSAY O B-TISSUE O B-TISSUE I-TISSUE O O O O O O B-DISEASE B-ORGANISM O O B-GENEPROD O O O O O O O O B-DISEASE B-ORGANISM O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O

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 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 ) .

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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 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 .

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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 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 .

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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 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 .

O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 .

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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 .

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 O O B-CELL_LINE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 .

O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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 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 .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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 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 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O 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) 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 .

O O O O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O 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 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 .

O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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 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 O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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 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 .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-EXP_ASSAY 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 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 .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 .

(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 .

O O O O O B-CELL_LINE O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 .

O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 .

O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 - 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 O O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 .

O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-TISSUE O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD 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 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 .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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 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 O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O 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-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 .

B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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 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 .

B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 ) .

B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

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 .

B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

B-CELL_LINE O O O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(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 the levels of β - catenin and RAS in DLD1 , WT - HCT116 , MT - HCT116 , HepG2 , and SW48 cells transfected with Myc - axin for 36

B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-CELL_LINE O O O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O

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