Figure 5In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . A Changes in body weights relative to day 0 were noted . The mean percentage changes in body weights for each cohort are plotted across all the days . Data information : n = 2 biological replicates with 5 mice per cohort were used . In panel A , across the entire experimental tenure of 14 days , data are presented as mean ± SD values for each cohort , averaged across both the replicates for all the five mice ( studied in the respective cohort ) . Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights across DMSO - treated and the respective peptide treated cohorts ( ns P > 0 . 05 ) . In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . B , After 14 days post peptide treatment , blood was collected from the animals by retro - orbital puncture and analysed for ( B ) alanine transaminase ( SGPT ) , alanine aminotransferase ( SGOT ) , alkaline phosphatase ( ALP ) Relative to the values obtained for the DMSO cohort , insignificant changes in serum levels of these parameters indicate that the peptide shows no significant hepatotoxic ( as seen in panel B ) Data information : biological replicates with 5 mice per cohort were used . In the panels B representative data for only one biological replicate across all the cohorts ( five mice per cohort ) are presented as mean ± SD values . P values were calculated in comparison to the DMSO - treated cohorts using Mann - Whitney rank sum test ( ns P > 0 . 05 ) . In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . C After 14 days post peptide treatment , blood was collected from the animals by retro - orbital puncture and analysed for ( C ) creatinine and blood urea nitrogen ( BUN ) levels . Relative to the values obtained for the DMSO cohort , insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects ( as seen in panel C ) . Data information : n = 2 biological replicates with 5 mice per cohort were used . In panels C , representative data for only one biological replicate across all the cohorts ( five mice per cohort ) are presented as mean ± SD values . For panels C , P values were calculated in comparison to the DMSO - treated cohorts using Mann - Whitney rank sum test ( ns P > 0 . 05 ) .

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

Figure 5In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. A Changes in body weights relative to day 0 were noted. The mean percentage changes in body weights for each cohort are plotted across all the days. Data information: n = 2 biological replicates with 5 mice per cohort were used. In panel A, across the entire experimental tenure of 14 days, data are presented as mean ± SD values for each cohort, averaged across both the replicates for all the five mice (studied in the respective cohort). Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights across DMSO-treated and the respective peptide treated cohorts (ns P > 0.05).In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. B, After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (B) alanine transaminase (SGPT), alanine aminotransferase (SGOT), alkaline phosphatase (ALP) Relative to the values obtained for the DMSO cohort, insignificant changes in serum levels of these parameters indicate that the peptide shows no significant hepatotoxic (as seen in panel B) Data information: biological replicates with 5 mice per cohort were used. In the panels B representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. C After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (C) creatinine and blood urea nitrogen (BUN) levels. Relative to the values obtained for the DMSO cohort, insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects (as seen in panel C). Data information: n = 2 biological replicates with 5 mice per cohort were used. In panels C, representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. For panels C, P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).


Figure 6Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . ( A ) Survival for symptoms were monitored . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . In panel A , percent survival was compared between untreated cohort ( N . T ) and the DMSO / peptide / ciprofloxacin - treated cohort using a two - way ANOVA test using Bonferroni correction ( ns P > 0 . 05 ; * P < 0 . 01 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively . Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . C - E Bacterial loads in S . Typhimurium infected organs , namely , ( C ) liver , ( D ) kidney and ( E ) spleen , were estimated . Data are presented as mean ± SD CFU / mL values for the bacterial organ loads isolated from seven mice in each cohort . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . In panels , ( C - E ) , Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively . Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . F - J Histopathology analyses with representative H & E - stained liver sections at different magnifications are shown for S . Typhimurium infected liver treated with various agents . K Mean pathological scoring from the tissue sections used in histopathology analyses in panels F - J . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . In panels K to assign histopathological scores , data are collected from five mice in each cohort , and the representative data for only one biological replicate are presented as mean ± SD values . Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively . For histopathology analyses in panels ( F - J ) except for uninfected and peptide - treated mice , pathology sections from infected or treated ( DMSO or ciprofloxacin ) mice showed aggregation of multiple inflammatory cells ( INF ) and edema ( E ) in liver parenchyma , central vein congestion ( C ) , distortion of hepatic portal vein ( HPV ) , hemorrhage ( H ) , necrosis ( N ) and pyknotic nuclei ( arrows in panels H , Scale bar : 50µm in panels ( F - J ) Action of the CP1 - WT peptide in treatment of A . baumannii intraperitoneal infections . L - N Bacterial loads in A . baumannii infected organs , namely , ( L ) liver , ( M ) kidney and ( N ) spleen , were estimated . Data are presented as mean ± SD CFU / mL values for the bacterial organ loads isolated from seven mice in each cohort . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . In panels ( L - N ) representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively .

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

Figure 6Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. (A) Survival for symptoms were monitored. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. In panel A, percent survival was compared between untreated cohort (N.T) and the DMSO/peptide/ciprofloxacin-treated cohort using a two-way ANOVA test using Bonferroni correction (ns P > 0.05; * P < 0.01). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. C-E Bacterial loads in S. Typhimurium infected organs, namely, (C) liver, (D) kidney and (E) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice in each cohort. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. In panels, (C-E), Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. F-J Histopathology analyses with representative H&E-stained liver sections at different magnifications are shown for S. Typhimurium infected liver treated with various agents. K Mean pathological scoring from the tissue sections used in histopathology analyses in panels F-J. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. In panels K to assign histopathological scores, data are collected from five mice in each cohort, and the representative data for only one biological replicate are presented as mean ± SD values. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively. For histopathology analyses in panels (F-J) except for uninfected and peptide-treated mice, pathology sections from infected or treated (DMSO or ciprofloxacin) mice showed aggregation of multiple inflammatory cells (INF) and edema (E) in liver parenchyma, central vein congestion (C), distortion of hepatic portal vein (HPV), hemorrhage (H), necrosis (N) and pyknotic nuclei (arrows in panels H, Scale bar: 50µm in panels (F-J)Action of the CP1-WT peptide in treatment of A. baumannii intraperitoneal infections. L-N Bacterial loads in A. baumannii infected organs, namely, (L) liver, (M) kidney and (N) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice in each cohort. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. In panels (L-N) representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.


Figure 1 ( F ) UMAP plot showing the expression levels of EMT markers . The magenta dotted lines indicate cluster 6 ( partial EMT ) .

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Figure 1(F) UMAP plot showing the expression levels of EMT markers. The magenta dotted lines indicate cluster 6 (partial EMT).


Figure 2 ( E ) , ( F ) Heatmap of DEGs in ROR1high and ROR1low cells in the S2 - VP10 xenograft ( E ) or PDO # 1 xenograft ( F ) .

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Figure 2(E), (F) Heatmap of DEGs in ROR1high and ROR1low cells in the S2-VP10 xenograft (E) or PDO#1 xenograft (F).


Figure 3 ( A ) Kaplan - Meier disease - specific survival curves based on ROR1 expression levels in PDAC patients from the TCGA database ( n = 154 ) . Data information : Graphs are presented as mean ± s . e . m . , * , p < 0 . 05 ; log - rank test ( A ) ( C ) FACS gating to sort ROR1high and ROR1low cells and the number of organoids or colonies ( n = 3 or n = 5 , biological replicates ) . Mouse cells expressing H - 2Kd / H - 2Dd were eliminated . Data information : Graphs are presented as mean ± s . e . m . , * , p < 0 . 05 ; two - sided t - test ( C ) ( G ) Representative images of tissue stained with H & E and AB / PAS , and for ROR1 in the PDO # 1 xenograft and of tumor derived from ROR1high or ROR1low cells in the PDO # 1 xenograft . Data information : Scale bars , 100 µm ( G ) .

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

Figure 3(A) Kaplan-Meier disease-specific survival curves based on ROR1 expression levels in PDAC patients from the TCGA database (n = 154) . Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; log-rank test (A)(C) FACS gating to sort ROR1high and ROR1low cells and the number of organoids or colonies (n = 3 or n = 5, biological replicates). Mouse cells expressing H-2Kd/H-2Dd were eliminated. Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; two-sided t-test (C)(G) Representative images of tissue stained with H&E and AB/PAS, and for ROR1 in the PDO#1 xenograft and of tumor derived from ROR1high or ROR1low cells in the PDO#1 xenograft. Data information: Scale bars, 100 µm (G).


Figure 4 ( H ) The expression level of AURKB in PANC - 1 cells transfected with control or ROR1 siRNA and in ROR1high or ROR1low cells from S2 - VP10 and PDO # 1 xenografts ( n = 3 ) . Data information : Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant . ( I ) Western blot analysis of ROR1 , AURKB , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA . ( J ) Treatment of PDO # 1 and S2 - VP10 organoids with DMSO ( vehicle ) or Aurora kinase inhibitors ( n = 3 , biological replicates ) . Tozasertib , pan - Aurora kinase inhibitor ; Barasertib , selective Aurora B kinase inhibitor . Representative images of organoids are shown . An area of 2 , 000 μm2 and more was identified as an organoid . The area of organoids is shown in the violin plot . Black or white solid lines indicate the median value for each violin . Data information : Scale bars 1 mm ( J ) Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant . ( K ) Organoid formation assay of ROR1high or ROR1low cells derived from S2 - VP10 EGFP ( control ) or S2 - VP10 AURKB - EGFP xenografts ( n = 3 , biological replicates ) . Representative images and the number of organoids are shown . Data information : Scale bars , 1 mm ( K ) . Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant . ( L ) Western blot analysis of ROR1 , AKT , phospho - AKT ( Ser473 ) , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA . ( N ) Western blot analysis of ROR1 , c - Myc , phospho - RB ( Ser780 ) , E2F1 , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA .

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

Figure 4(H) The expression level of AURKB in PANC-1 cells transfected with control or ROR1 siRNA and in ROR1high or ROR1low cells from S2-VP10 and PDO#1 xenografts (n = 3). Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.(I) Western blot analysis of ROR1, AURKB, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.(J) Treatment of PDO#1 and S2-VP10 organoids with DMSO (vehicle) or Aurora kinase inhibitors (n = 3, biological replicates). Tozasertib, pan-Aurora kinase inhibitor; Barasertib, selective Aurora B kinase inhibitor. Representative images of organoids are shown. An area of 2,000 μm2 and more was identified as an organoid. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars 1 mm (J) Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.(K) Organoid formation assay of ROR1high or ROR1low cells derived from S2-VP10 EGFP (control) or S2-VP10 AURKB-EGFP xenografts (n = 3, biological replicates). Representative images and the number of organoids are shown. Data information: Scale bars, 1 mm (K). Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.(L) Western blot analysis of ROR1, AKT, phospho-AKT (Ser473), and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.(N) Western blot analysis of ROR1, c-Myc, phospho-RB (Ser780), E2F1, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.


Figure 5 ( C ) Tumor growth curve of the four groups ( control , Dox only , gemcitabine only , and Dox + gemcitabine ) ( n = 8 ) . The endpoint for each group is shown by a slash . Data information : Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 .

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Figure 5(C) Tumor growth curve of the four groups (control, Dox only, gemcitabine only, and Dox + gemcitabine) (n = 8). The endpoint for each group is shown by a slash. Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05.


Figure 6 ( A ) - ( C ) Representative images of ROR1 , mCherry , and aSMA staining in the primary tumor ( pancreas ) ( A ) and metastatic lesions in the lung ( B ) and mesenteric lymph node ( C ) after orthotopic transplantation of S2 - VP10 - mCherry cells into the pancreas of BRJ mice . Data information : Scale bars , 100 µm ( D ) , ( E ) Representative images of S2 - 013 orthotopic xenografts stained with H & E and for ROR1 in the pancreas ( D ) and mesenteric lymph node ( E ) . Data information : Scale bars , 100

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

Figure 6(A)-(C) Representative images of ROR1, mCherry, and aSMA staining in the primary tumor (pancreas) (A) and metastatic lesions in the lung (B) and mesenteric lymph node (C) after orthotopic transplantation of S2-VP10-mCherry cells into the pancreas of BRJ mice. Data information: Scale bars, 100 µm(D), (E) Representative images of S2-013 orthotopic xenografts stained with H&E and for ROR1 in the pancreas (D) and mesenteric lymph node (E). Data information: Scale bars, 100 µm


Figure 7 ( A ) , H3K4me1 , K3K4me3 , H3K27ac , and open chromatin profiles around the ROR1 gene by CUT & RUN and ATAC - sequencing in cultured S2 - VP10 cells ( A ) The ROR1 promoter ( blue - shaded boxes ) and enhancer elements ( green - shaded box ) of ROR1 are indicated .

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Figure 7(A), H3K4me1, K3K4me3, H3K27ac, and open chromatin profiles around the ROR1 gene by CUT&RUN and ATAC-sequencing in cultured S2-VP10 cells (A) The ROR1 promoter (blue-shaded boxes) and enhancer elements (green-shaded box) of ROR1 are indicated.


Figure 8 ( E ) YAP occupancy at the ROR1 enhancer as determined by ChIP - qPCR . The known YAP targets , CTGF and CYR61 promoters , were tested as positive controls , and ROR1 intron 1 and AFM promoter were tested as negative controls ( n = 4 , biological replicates ) . ( G ) Western blot analysis of YAP , TAZ , ROR1 , and β - actin in S2 - VP10 cells transfected with control or YAP1 / WWTR1 siRNA . ( I ) Relative expression of ROR1 and YAP target genes ( CTGF and CYR61 ) in S2 - VP10 cells treated with DMSO or verteporfin ( VP ) ( n = 3 , biological replicates ) . mRNA levels are normalized to that of RPS18 . ( M ) Relative luciferase activity using the ROR1 reporter in S2 - VP10 cells treated with DMSO or BET inhibitor ( JQ1 ) ( n = 3 , biological replicates ) . Data information : Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( M ) ( O ) , Treatment of PDO # 1 and S2 - VP10 organoids with DMSO or JQ1 ( n = 3 , biological replicates ) . Relative expression of ROR1 ( O ) , mRNA levels are normalized to that of RPS18 . Data information : Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( O ) ( P ) Treatment of PDO # 1 and S2 - VP10 organoids with DMSO or JQ1 ( n = 3 , biological replicates ) . representative images of organoids , and organoid area ( P ) are shown . The area of organoids is shown in the violin plot . Black or white solid lines indicate the median value for each violin . Data information : Scale bars , 1 mm ( P ) . Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( P ) .

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

Figure 8(E) YAP occupancy at the ROR1 enhancer as determined by ChIP-qPCR. The known YAP targets, CTGF and CYR61 promoters, were tested as positive controls, and ROR1 intron 1 and AFM promoter were tested as negative controls (n = 4, biological replicates).(G) Western blot analysis of YAP, TAZ, ROR1, and β-actin in S2-VP10 cells transfected with control or YAP1/WWTR1 siRNA.(I) Relative expression of ROR1 and YAP target genes (CTGF and CYR61) in S2-VP10 cells treated with DMSO or verteporfin (VP) (n = 3, biological replicates). mRNA levels are normalized to that of RPS18.(M) Relative luciferase activity using the ROR1 reporter in S2-VP10 cells treated with DMSO or BET inhibitor (JQ1) (n = 3, biological replicates). Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (M)(O), Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). Relative expression of ROR1 (O), mRNA levels are normalized to that of RPS18. Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (O)(P) Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). representative images of organoids, and organoid area (P) are shown. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars, 1 mm (P). Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (P).


Figure 1A BAK - 6H permeabilising activity is exacerbated by cBID . BAK - 6H ( mBAK ∆ C21 - 6H ) was loaded at the indicated concentrations onto Ni - NTA liposomes ( 5 μM ) prior to the addition of cBID . The release of carboxyfluorescein was measured over time . Results representative of at least 3 independent experiments . B BAK - 6H oligomerises on liposomes induced by cBID . Liposomes were incubated with BAK - 6H ( 150 nM ) and the indicated concentration of cBID or a BID BH3 peptide for 60 min . Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN - PAGE . In parallel , samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran , normalised to total fluorescence of detergent permeabilised liposomes . Results representative of 3 independent experiments . C BAK - 6H oligomerisation on liposomes is blocked by BCL - XL . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( WT , 90 nM ) or cBID M97A ( 90 nM ) that has reduced affinity for BCL - XL , in the presence or absence of BCL - XL for 60 mins prior to analysis of BAK oligomerisation by BN - PAGE . Results representative of 2 independent experiments . Liposome permeabilisation was monitored with the indicated combinations of cBID ( 90 nM ) , BAK - 6H ( 150 nM ) and BCL - XL ( 1 μM ) . Data is presented as mean of 2 independent experiments with percentage release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50 % of detergent - lysis maximum . D BAK - 6H oligomerisation on liposomes involves its BH3 domain . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( 90 nM ) . Antibody recognising the BAK BH3 domain ( 4B5 ) or α1 - α2 loop ( 7D10 ) , as shown on the structure of BAK ( PDB : 2IMS ) , were added ( 2 . 4 μg / μl ) either prior to ( pre ) or after ( post ) incubation with cBID for 60 mins . BAK : Ab complex is indicated ( arrowhead ) . 7D10 which recognises the activated form of mBAK ( Iyer et al . , 2016 ) , bound to and gel - shifted BAK when added either before or after activation with cBID . In contrast , 4B5 failed to appreciably gel - shift inactive BAK ( lanes 3 and 5 ) or BAK that was already activated ( lane 6 ) , but gel - shifted BAK when present during cBID activation ( lane 4 ) . Liposome permeabilisation was assessed after 20 min incubation with cBID ( 90 nM ) , BAK - 6H ( 150 nM ) and antibodies 4B5 ( 2 . 5 μg ) or 7D10 ( 2 . 5 μg ) . Data is presented as mean of 2 independent experiments with percentage release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50 % of detergent - lysis maximum .

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

Figure 1A BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 μM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least 3 independent experiments.B BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of 3 independent experiments.C BAK-6H oligomerisation on liposomes is blocked by BCL-XL. Liposomes were incubated with BAK-6H (150 nM) together with cBID (WT, 90 nM) or cBID M97A (90 nM) that has reduced affinity for BCL-XL, in the presence or absence of BCL-XL for 60 mins prior to analysis of BAK oligomerisation by BN-PAGE. Results representative of 2 independent experiments. Liposome permeabilisation was monitored with the indicated combinations of cBID (90 nM), BAK-6H (150 nM) and BCL-XL (1 μM). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.D BAK-6H oligomerisation on liposomes involves its BH3 domain. Liposomes were incubated with BAK-6H (150 nM) together with cBID (90 nM). Antibody recognising the BAK BH3 domain (4B5) or α1-α2 loop (7D10), as shown on the structure of BAK (PDB:2IMS), were added (2.4 μg/μl) either prior to (pre) or after (post) incubation with cBID for 60 mins. BAK:Ab complex is indicated (arrowhead). 7D10 which recognises the activated form of mBAK (Iyer et al., 2016), bound to and gel-shifted BAK when added either before or after activation with cBID. In contrast, 4B5 failed to appreciably gel-shift inactive BAK (lanes 3 and 5) or BAK that was already activated (lane 6), but gel-shifted BAK when present during cBID activation (lane 4). Liposome permeabilisation was assessed after 20 min incubation with cBID (90 nM), BAK-6H (150 nM) and antibodies 4B5 (2.5 μg) or 7D10 (2.5 μg). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


Figure 3B BAK , or BAK with cysteine introduced at the indicated positions ( on a BAK ∆ Cys background ) were stably expressed in Bax - / - Bak - / - MEFs and assess for BAK expression by immunoblotting ( inset panel ; * indicates non - specific band . ) and apoptotic activity in response to etoposide treatment . C E24 and R169 are proximal in inactive BAK on mitochondria . Mitochondria - enriched membrane fractions from cells were incubated with oxidant ( CuPhe ) and induced intramolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE . D E24 and R169 dissociate during BAK activation . Mitochondria - enriched membrane fractions from cells were incubated with cBID ( 100 nM ) prior to oxidant ( CuPhe ) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

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

Figure 3B BAK, or BAK with cysteine introduced at the indicated positions (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and assess for BAK expression by immunoblotting (inset panel; * indicates non-specific band.) and apoptotic activity in response to etoposide treatment.C E24 and R169 are proximal in inactive BAK on mitochondria. Mitochondria-enriched membrane fractions from cells were incubated with oxidant (CuPhe) and induced intramolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.D E24 and R169 dissociate during BAK activation. Mitochondria-enriched membrane fractions from cells were incubated with cBID (100 nM) prior to oxidant (CuPhe) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


Figure 4C Bax - / - Bak - / - MEFs expressing the indicated BAK variants were treated with doxycycline ( Dox , 3 h ) , incubated for a further 24 h in the presence or absence of BH3 - mimetic compounds , and cell death was assessed by PI uptake . D Mutation of BH4 promotes BAK activating conformation change . Cells were treated with doxycycline ( 3 h ) to induce BAK expression followed by incubation with BH3 - mimetics where indicated ( for 2 h ) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK ( G3172 ) .

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Figure 4C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake.D Mutation of BH4 promotes BAK activating conformation change. Cells were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172).


Figure 5D BAK wild - type , or BAK with an engineered α1 - α2 loop thrombin cleavage site were stably expressed in Bax - / - Bak - / - MEFs . Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline ( CuPhe ) to induce disulphide - linkage . Samples were run on SDS - PAGE under non - reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N - terminus upstream of the thrombin cleavage site ( aa23 - 38 ) or an antibody recognising the BH3 domain ( 4B5 ) downstream of the cleavage site . Schematic indicates the nature of the BAK protein detected by each antibody . Disulphide - linkage indicated by a hashed line . Note that the N - terminal portion of thrombin cleaved BAK could not be detected with the N - terminal antibody unless it was intramolecularly disulphide - linked . * cBID ( 100 nM ) was added to activate BAK during thrombin cleavage . Data representative of two independent experiments . E Cleavage of the BAK α1 - α2 loop potentiates MOMP . Membrane fractions from Bax - / - Bak - / - MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID ( 10 nM ) for the indicated times . Membrane ( P ) and supernatant ( S ) fractions were separated and immunoblotted for cytochrome c or BAK 4B5 .

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

Figure 5D BAK wild-type, or BAK with an engineered α1-α2 loop thrombin cleavage site were stably expressed in Bax-/-Bak-/- MEFs. Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline (CuPhe) to induce disulphide-linkage. Samples were run on SDS-PAGE under non-reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N-terminus upstream of the thrombin cleavage site (aa23-38) or an antibody recognising the BH3 domain (4B5) downstream of the cleavage site. Schematic indicates the nature of the BAK protein detected by each antibody. Disulphide-linkage indicated by a hashed line. Note that the N-terminal portion of thrombin cleaved BAK could not be detected with the N-terminal antibody unless it was intramolecularly disulphide-linked. *cBID (100 nM) was added to activate BAK during thrombin cleavage. Data representative of two independent experiments.E Cleavage of the BAK α1-α2 loop potentiates MOMP. Membrane fractions from Bax-/-Bak-/- MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID (10 nM) for the indicated times. Membrane (P) and supernatant (S) fractions were separated and immunoblotted for cytochrome c or BAK 4B5.


Figure 1A ) H & E and immunohistochemistry reveal that β - catenin accumulates in tumors emerging in one - year - old female miR - 424 ( 322 ) / 503 - / - mice . The panel is a representative image of n = 5 independent tumors . The scale bar indicates 100 micrometers ( µM ) . B ) Bar graphic showing that human breast cancers with deletion of the miR - 424 / 503 locus have higher protein levels of β - catenin ( RPPA ) . P - value is calculated based on one - sided Wilcoxon rank - sum test . In the box - plots in B and C the relative expression of β - catenin in the RPPA data from TCGA is shown . The yellow line indicates the median and the bars indicate the 75th percentile ( Q3 ) + 1 . 5 interquartile range ( IQR ) . C ) The bar graph shows that human breast cancers with low expression levels of miR - 424 and miR - 503 have higher protein levels of β - catenin . P - value is calculated based on one - sided Wilcoxon rank - sum test .

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

Figure 1A) H&E and immunohistochemistry reveal that β-catenin accumulates in tumors emerging in one-year-old female miR-424(322)/503-/- mice. The panel is a representative image of n=5 independent tumors. The scale bar indicates 100 micrometers (µM).B) Bar graphic showing that human breast cancers with deletion of the miR-424/503 locus have higher protein levels of β-catenin (RPPA). P-value is calculated based on one-sided Wilcoxon rank-sum test. In the box-plots in B and C the relative expression of β-catenin in the RPPA data from TCGA is shown. The yellow line indicates the median and the bars indicate the 75th percentile (Q3)+1.5 interquartile range (IQR).C) The bar graph shows that human breast cancers with low expression levels of miR-424 and miR-503 have higher protein levels of β-catenin. P-value is calculated based on one-sided Wilcoxon rank-sum test.


Figure 2B ) The bar graphs show the number of PROCR + in 1 - year - old female WT and miR - 424 ( 322 ) / 503 KO mice during diestrus . The error bars show the standard deviation . E ) The panel shows the lack of induction of bona fide WNT targets in miR - over expressing cells from panel C compared with EV control . UT indicates samples untreated with WNT3 .

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Figure 2B) The bar graphs show the number of PROCR+ in 1-year-old female WT and miR-424(322)/503 KO mice during diestrus. The error bars show the standard deviation.E) The panel shows the lack of induction of bona fide WNT targets in miR-over expressing cells from panel C compared with EV control. UT indicates samples untreated with WNT3.


Figure 3D ) Western blot of LRP6 and TBLR1 in the miR - cluster - Dox - MCF - 10A model upon upregulation of miR - cluster with 100 ng / ml of doxycycline for the specified number of days . The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β - actin . The expression level of miR - 424 and miR - 503 is indicated in the bar graph below the blots . UN indicates samples untreated with doxycycline . The error bars show the standard deviation . F ) Western blot of LRP6 and TBLR1 expression in WT and miR - 424 / 503 - KO basal cell - derived organoids after 14 days in culture with and without Wnt3a ( 100ng / ml ) . The arrow shows the correct TBLR1 band . G ) IHC staining showing that LRP6 accumulates in the epithelium of female miR - 424 ( 322 ) / 503 - / - mice ( 1 - year - old ) . The scale bar indicates 100 micrometers ( µM ) .

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

Figure 3D) Western blot of LRP6 and TBLR1 in the miR-cluster-Dox-MCF-10A model upon upregulation of miR-cluster with 100 ng/ml of doxycycline for the specified number of days. The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β-actin. The expression level of miR-424 and miR-503 is indicated in the bar graph below the blots. UN indicates samples untreated with doxycycline. The error bars show the standard deviation.F) Western blot of LRP6 and TBLR1 expression in WT and miR-424/503-KO basal cell-derived organoids after 14 days in culture with and without Wnt3a (100ng/ml). The arrow shows the correct TBLR1 band.G) IHC staining showing that LRP6 accumulates in the epithelium of female miR-424(322)/503-/- mice (1-year-old). The scale bar indicates 100 micrometers (µM).


Figure 4A ) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR - cluster - Dox - MCF - 10A cells after the miR - 424 / 503 cluster is induced with 100ng / ml of dox for three days . A CDC25A - binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls . The error bars show the standard deviation . B ) The WT - blot shows the expression of LRP6 upon upregulation of miR - 424 / 503 in miR - cluster - Dox - MCF - 10A cells ( Dox + ) when one or both of the conserved microRNA binding sites ( # 1 and # 3 ) are blocked by target protection . C ) The WT - blot shows the expression of LRP6 and β - catenin in cells in miR - cluster - Dox - MCF - 10A cells cultured in the presence of Dox and with and without WNT3a ligand ( 100ng / ml for 6 hours ) when the microRNA binding sites # 1 and # 3 are blocked by target protection .

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

Figure 4A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation.B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection.C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.


Figure 1Nurr1 ( N ) and Foxa2 ( F ) synergistically downregulate SGK1 expression in cultured glia . ( A ) Up - and downregulated genes in the microarray data for the cultured glia transduced with N + F ( vs . mock - transduced control ) . Sgk1 is marked with an arrow in the top downregulated genes . Effect of N and F on SGK1 expression in glial cells shown in the microarray ( B ) , RNA - seq ( C ) , Data in ( B and C ) represent values of fold change ( FC ) or read count ( RC ) with color intensities . n = 3 . One - way ANOVA . Data are represented as mean ± SEM . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * . Effect of N and F on SGK1 expression in glial cells shown in the RT - PCR analyses . One - way ANOVA . Data are represented as mean ± SEM . Significantly different at p = 0 . 0493 * , 0 . 0092 * * , 0 . 0009 * * * . Downregulation of SGK1 is responsible for N + F function to inhibit NFκB - mediated pro - inflammatory cytokine expression . ( E ) WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation WB analysis exhibiting N + F - induced inhibition of NFκB intracellular signaling in cultured VM - glia . The N + F - mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation ( E ) and thus blocking the release of NFκB from the NFκB - IκB inhibitory complex ( F ) . N + F - mediated downregulation of NFκB signaling ( phosphorylation of p65 , p - p65 , G ) were alleviated by forced SGK1 expression in the VM - glial cultures . H ) N + F - mediated downregulation of pro - inflammatory cytokine expression ( IL - 1β , TNFα , H ) were alleviated by forced SGK1 expression in the VM - glial cultures . ( I ) SGK1 inhibition blocks glial NFκB signaling . Data are represented as mean ± SEM . n = 4 - 6 . One - way ANOVA . Significantly different at p = 0 . 002 * , 0 . 00004 * * , 0 . 00001 # , 3 . 01E - 10 # # , 3 . 78E - 06 + , 1 . 59E - 07 + + in graph I . Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition Glial pro - inflammatory cytokine expression regulated by SGK1 inhibition ( J - M ) and overexpression ( N ) .

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

Figure 1Nurr1 (N) and Foxa2 (F) synergistically downregulate SGK1 expression in cultured glia. (A) Up- and downregulated genes in the microarray data for the cultured glia transduced with N+F (vs. mock-transduced control). Sgk1 is marked with an arrow in the top downregulated genes. Effect of N and F on SGK1 expression in glial cells shown in the microarray (B), RNA-seq (C), Data in (B and C) represent values of fold change (FC) or read count (RC) with color intensities. n=3. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.  Effect of N and F on SGK1 expression in glial cells shown in the RT-PCR analyses. One-way ANOVA. Data are represented as mean ± SEM. Significantly different at p= 0.0493*, 0.0092**, 0.0009***.  Downregulation of SGK1 is responsible for N+F function to inhibit NFκB-mediated pro-inflammatory cytokine expression. (E) WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation  WB analysis exhibiting N+F-induced inhibition of NFκB intracellular signaling in cultured VM-glia. The N+F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation (E) and thus blocking the release of NFκB from the NFκB-IκB inhibitory complex (F).  N+F-mediated downregulation of NFκB signaling (phosphorylation of p65, p-p65, G) were alleviated by forced SGK1 expression in the VM-glial cultures.  H) N+F-mediated downregulation of pro-inflammatory cytokine expression (IL-1β, TNFα, H) were alleviated by forced SGK1 expression in the VM-glial cultures.  (I) SGK1 inhibition blocks glial NFκB signaling. Data are represented as mean ± SEM. n=4-6. One-way ANOVA. Significantly different at p=0.002*, 0.00004**, 0.00001#, 3.01E-10##, 3.78E-06+, 1.59E-07++ in graph I.  Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition  Glial pro-inflammatory cytokine expression regulated by SGK1 inhibition (J-M) and overexpression (N).


Figure 2 In the RNA - seq data ( GSE145490 , normalized read count ( RC ) > 1 , > 2 . 6 - fold , 3 , 000 genes ) , genes downregulated in the VM - glia treated with the SGK1 inhibitor GSK - 650394 vs the vehicle ( DMSO ) - treated control were analyzed . A and B , Top gene ontologies ( GOs ) and KEGG pathways of the genes downregulated in VM - glia by the SGK1 inhibitor treatment . The purple bar indicates the number of genes under the designated GO term / KEGG pathway . The red bar indicates the p - value , and the negative log of the p - value ( bottom ) is plotted on the X - axis . C , Gene set enrichment analysis ( GESA ) for inflammatory and immune responses . D , Scatterplots for the differentially expressed genes ( DEGs ) highlighting the inflammatory / immune genes that are most significantly downregulated in the inhibiter - treated cultures . E , Heatmap for selected inflammatory genes in the SGK1 inhibitor - treated and - untreated glial cultures . Data represent the RC values ( inside box ) and log2 [ SGK1 inhibitor - treated / control ] ( color intensity ) . F , Microglia and astrocyte reactivation - specific marker expressions in the RNA - seq data .

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

Figure 2 In the RNA-seq data (GSE145490, normalized read count (RC)>1, >2.6-fold, 3,000 genes), genes downregulated in the VM-glia treated with the SGK1 inhibitor GSK-650394 vs the vehicle (DMSO)-treated control were analyzed. A and B, Top gene ontologies (GOs) and KEGG pathways of the genes downregulated in VM-glia by the SGK1 inhibitor treatment. The purple bar indicates the number of genes under the designated GO term/KEGG pathway. The red bar indicates the p-value, and the negative log of the p-value (bottom) is plotted on the X-axis. C, Gene set enrichment analysis (GESA) for inflammatory and immune responses. D, Scatterplots for the differentially expressed genes (DEGs) highlighting the inflammatory/immune genes that are most significantly downregulated in the inhibiter-treated cultures.  E, Heatmap for selected inflammatory genes in the SGK1 inhibitor-treated and -untreated glial cultures. Data represent the RC values (inside box) and log2[SGK1 inhibitor-treated/control] (color intensity).  F, Microglia and astrocyte reactivation-specific marker expressions in the RNA-seq data.


Figure 3 mRNA expression of the NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , and Caspase1 . Decreased expression of the components shown in the RNA - seq Data are expressed as mean ± SEM . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + + B , mRNA expression of the NLRP3 - inflammasome components Nlrp3 , Pycard ( Asc ) , and Caspase1 . Decreased expression of the components shown in the q - PCR analyses in other independent VM - glial cultures treated with the inhibitor ( GSK - 650394 ) and sh - Sgk1 and si - Sgk1 ( vs . DMSO , sh - control , and si - control , respectively ) ( B ) . n = 3 ; Student ' s t - test . Data are expressed as mean ± SEM . Significantly different at p = 0 . 0018 * , 0 . 0405 * * , 0 . 0039 * * * , 0 . 0002 # , 0 . 0033 # # , 0 . 0099 # # # , 0 . 0001 # # # # , 0 . 0056 + , 0 . 0398 + + , 0 . 0067 + + + in graph B . C , Immunoblot analysis to assess NLRP3 inflammasome activation . To activate the inflammasome pathway , VM - derived glial cells were treated with LPS ( 0 . 25 µg / ml , 3h ) and then ATP ( 2 . 5 mM , 30 min ) in the presence or absence of the SGK1 inhibitor GSK - 650394 . Two days later , intracellular and released levels of pro - and activated caspase - 1 and IL - 1β proteins were determined in the culture media and cell lysates , respectively . D , Released level of IL - 1β ( activated ) was further quantified using ELISA . n = 3 ; ANOVA . Significantly different at p = 0 . 0002 # , 0 . 0012 * , 0 . 0004 * * in graph D . Expression of CGAS - STING pathway genes in the RNA - seq data . The graph represents log2 RC ratios of [ GSK - 650394 - treated / DMSO - treated control ] . G , WB analysis demonstrating suppression of CGAS - STING signaling by the SGK1 inhibitor in VM - glial cultures . Representative blots are shown on the left . The levels of the active forms of CGAS - STING signaling molecules are quantified from 5 - 10 independent blots ( right ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 0 . 001 # , 0 . 014 * ( CGAS ) , 0 . 00004 # , 0 . 0002 * ( STING ) , 0 . 017 # , 0 . 037 * ( pTBK1 ) , 0 . 009 # , 0 . 045 * ( pIRF3 ) , 0 . 012 # , 0 . 036 * ( pP65 ) in graph G . H , Expression of Ifnb mRNA , a final product of the CGAS - STING pathway , determined by real - time PCR analyses . Data are represented as mean ± SEM . n = 3 ; student ' s t - test . Significantly different at p = 0 . 0459 # , 0 . 0305 * in graph H .

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

Figure 3 mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the RNA-seq Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++  B, mRNA expression of the NLRP3-inflammasome components Nlrp3, Pycard (Asc), and Caspase1. Decreased expression of the components shown in the q-PCR analyses in other independent VM-glial cultures treated with the inhibitor (GSK-650394) and sh-Sgk1 and si-Sgk1 (vs. DMSO, sh-control, and si-control, respectively) (B). n=3; Student's t-test. Data are expressed as mean ± SEM. Significantly different at p=0.0018*, 0.0405**, 0.0039***, 0.0002#, 0.0033##, 0.0099###, 0.0001####, 0.0056+, 0.0398++, 0.0067+++ in graph B.  C, Immunoblot analysis to assess NLRP3 inflammasome activation. To activate the inflammasome pathway, VM-derived glial cells were treated with LPS (0.25 µg/ml, 3h) and then ATP (2.5 mM, 30 min) in the presence or absence of the SGK1 inhibitor GSK-650394. Two days later, intracellular and released levels of pro- and activated caspase-1 and IL-1β proteins were determined in the culture media and cell lysates, respectively.  D, Released level of IL-1β (activated) was further quantified using ELISA. n=3; ANOVA. Significantly different at p=0.0002#, 0.0012*, 0.0004** in graph D.  Expression of CGAS-STING pathway genes in the RNA-seq data. The graph represents log2 RC ratios of [GSK-650394-treated/DMSO-treated control].  G, WB analysis demonstrating suppression of CGAS-STING signaling by the SGK1 inhibitor in VM-glial cultures. Representative blots are shown on the left. The levels of the active forms of CGAS-STING signaling molecules are quantified from 5-10 independent blots (right). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.001#, 0.014* (CGAS), 0.00004#, 0.0002* (STING), 0.017#, 0.037* (pTBK1), 0.009#, 0.045* (pIRF3), 0.012#, 0.036* (pP65) in graph G.  H, Expression of Ifnb mRNA, a final product of the CGAS-STING pathway, determined by real-time PCR analyses. Data are represented as mean ± SEM. n=3; student's t-test. Significantly different at p=0.0459#, 0.0305* in graph H.


Figure 4 Ppargc1a ( PGC1α ) mRNA expression upregulated in VM - glia treated with the SGK1 inhibitors in the RNA - seq data Data are represented as mean ± SEM . n = 3 ; Student ' s t - test . Significantly different at p = 0 . 0011 # , 0 . 0499 * in graph B . B , Ppargc1a ( PGC1α ) mRNA expression upregulated in VM - glia treated with the SGK1 inhibitors in the q - PCR analysis Data are represented as mean ± SEM . n = 3 ; Student ' s t - test . Significantly different at p = 0 . 0011 # , 0 . 0499 * in graph B . C , Immunoblot for PGC1α protein expression . Data are represented as mean ± SEM . n = 3 . Student ' s t - test . Significantly different at p = 0 . 0077 * in graph C . D , Mitochondrial biogenesis estimated by Mitotimer reporter gene expression in cultured VM - glia . Green fluorescence indicates newly synthesized mitochondria , and red fluorescence indicates old or damaged mitochondria . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 3 . 78E - 08 * , 0 . 00003 * * in graph D . E , Mitochondrial ROS levels estimated using the MitoSox red probe . Fluorescence intensity was measured using ImageJ , and ROS levels are presented as mean fluorescent intensity ( MFI ) . Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP ( 2µM ) + H2O2 ( 500 µM ) for 4hr . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 0 . 0013 * , 0 . 0273 # in graph E . F , Mitochondrial membrane potential ( JC - 1 ) . The red fluorescence ( JC - 1 Aggregate ) indicates disrupted mitochondrial membrane potential , and green fluorescence ( JC - 1 Monomer , insets ) indicates intact potential . The inset images were taken from the same microscopic fields of the respective JC - 1 aggregate images . Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP ( 2µM ) + H2O2 ( 500 µM ) for 4hr . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± SEM . One - way ANOVA . Significantly different at p = 0 . 0006 * , 0 . 0093 # in graph F . G , Intracellular levels of ATP , an indicator of mitochondrial oxidative phosphorylation . Data are expressed as mean ± SEM . n = 3 cultures ; one - way ANOVA . Significantly different at p = 0 . 0009 * , 0 . 00004 * * , 0 . 04913 # , 0 . 0024 + in graph G . Glial cell senescence rescued by SGK1 inhibition . ( H ) Expression of genes associated with cell senescence in the RNA - seq data ( VM - glia with vs . without GSK - 650394 treatment ) . ( I ) Intracellular ROS levels estimated by DCF - DA . Data are represented as mean ± SEM . n = 3 ; one - way ANOVA . Significantly different at p = 0 . 0074 * , 0 . 012 # in graph I . ( J ) Senescence - associated β - galactosidase ( SA - β - gal ) activity . Data are represented as mean ± SEM . n = 3 ; one - way ANOVA . Significantly different at p = 2 . 05E - 06 * , 0 . 0042 # , 0 . 0006 + in graph J . ( K ) Immunoblots for proteins associated with cell senescence . Significantly different at p = 0 . 008 * , 0 . 001 # ( TNFA ) , 0 . 01 * , 0 . 009 # ( HMGB1 ) , 7 . 32E - 10 * , 1 . 23E - 8 # ( IL6 ) , 9 . 62E - 11 * , 1 . 01E - 5 # ( MMP3 ) , 0 . 001 * , 5 . 72E - 4 # ( LAMIN B ) in graph K . ( L ) IL - 6 levels secreted from cultured VM - glia . The cytokine levels were estimated using ELISA of the media from the VM - glia with and without the SGK1 inhibitor . Data are represented as mean ± SEM . n = 3 cultures . One - way ANOVA . Significantly different at p = 0 . 0028 * , 0 . 0062 # in graph L . ( M ) Glial cell viability following treatment with senolytic compounds ( azithromycin , fisetin ) . Data are represented as mean ± SEM . n = 3 ; Student ' s t - test . Significantly different at p = 0 . 0073 * , 0 . 0018 * * , 0 . 0286 # , 0 . 0269 # # , 0 . 0045 # # # in graph M .

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

Figure 4 Ppargc1a (PGC1α) mRNA expression upregulated in VM-glia treated with the SGK1 inhibitors in the RNA-seq data Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0011#, 0.0499* in graph B.  B, Ppargc1a (PGC1α) mRNA expression upregulated in VM-glia treated with the SGK1 inhibitors in the q-PCR analysis Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0011#, 0.0499* in graph B.  C, Immunoblot for PGC1α protein expression. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0077* in graph C. D, Mitochondrial biogenesis estimated by Mitotimer reporter gene expression in cultured VM-glia. Green fluorescence indicates newly synthesized mitochondria, and red fluorescence indicates old or damaged mitochondria. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=3.78E-08*, 0.00003** in graph D. E, Mitochondrial ROS levels estimated using the MitoSox red probe. Fluorescence intensity was measured using ImageJ, and ROS levels are presented as mean fluorescent intensity (MFI). Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0013*, 0.0273# in graph E. F, Mitochondrial membrane potential (JC-1). The red fluorescence (JC-1 Aggregate) indicates disrupted mitochondrial membrane potential, and green fluorescence (JC-1 Monomer, insets) indicates intact potential. The inset images were taken from the same microscopic fields of the respective JC-1 aggregate images. Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0006*, 0.0093# in graph F. G, Intracellular levels of ATP, an indicator of mitochondrial oxidative phosphorylation. Data are expressed as mean ± SEM. n=3 cultures; one-way ANOVA. Significantly different at p= 0.0009*, 0.00004**, 0.04913#, 0.0024+in graph G.  Glial cell senescence rescued by SGK1 inhibition. (H) Expression of genes associated with cell senescence in the RNA-seq data (VM-glia with vs. without GSK-650394 treatment).  (I) Intracellular ROS levels estimated by DCF-DA. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=0.0074*, 0.012# in graph I.  (J) Senescence-associated β-galactosidase (SA-β-gal) activity. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=2.05E-06*, 0.0042#, 0.0006+ in graph J.  (K) Immunoblots for proteins associated with cell senescence. Significantly different at p=0.008*, 0.001# (TNFA), 0.01*, 0.009# (HMGB1), 7.32E-10*, 1.23E-8# (IL6), 9.62E-11*, 1.01E-5# (MMP3), 0.001*, 5.72E-4# (LAMIN B) in graph K.  (L) IL-6 levels secreted from cultured VM-glia. The cytokine levels were estimated using ELISA of the media from the VM-glia with and without the SGK1 inhibitor. Data are represented as mean ± SEM. n=3 cultures. One-way ANOVA. Significantly different at p=0.0028*, 0.0062# in graph L.  (M) Glial cell viability following treatment with senolytic compounds (azithromycin, fisetin). Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0073*,0.0018**, 0.0286#, 0.0269##, 0.0045### in graph M.


Figure 5B , Western blot analysis for detecting SNCA oligomers . Data are represented as mean ± SEM . n = 4 ; one - way ANOVA . Significantly different at p = 2 . 5E - 07 * , 1 . 65E - 07 * * , 0 . 022 # , 0 . 029 # # , 0 . 046 + , 0 . 015 + + , 0 . 0136 & , 0 . 0016 & & in graph B . C , Immunocytochemical detection of p - 129 - SNCA , associated with SNCA aggregation . Data are represented as mean ± SEM . n = 6 . Significantly different at p = 0 . 0363 * in graph C . E , Bi - FC - based assessment of the inter - neuronal propagation and aggregation of SNCA . Bi - FC + inclusions ( puncta ) were counted in E . Data are represented as mean ± SEM . n = 5 . Significantly different at p = 0 . 0446 * in graph E . VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , and the conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation were assessed by WB VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , and the conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation and p - 129 - SNCA immunocytochemical analyses . Data are represented as mean ± SEM . n = 3 . Significantly different at p = 0 . 00003 * , 0 . 0003 * * , 0 . 0012 * * * , 0 . 00004 * * * * , 0 . 0321 # in graph G and p = 0 . 0052 * in graph H .

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

Figure 5B, Western blot analysis for detecting SNCA oligomers. Data are represented as mean ± SEM. n=4; one-way ANOVA. Significantly different at p=2.5E-07*, 1.65E-07**, 0.022#, 0.029##, 0.046+, 0.015++, 0.0136&, 0.0016&& in graph B. C, Immunocytochemical detection of p-129-SNCA, associated with SNCA aggregation. Data are represented as mean ± SEM. n=6. Significantly different at p=0.0363* in graph C.  E, Bi-FC-based assessment of the inter-neuronal propagation and aggregation of SNCA. Bi-FC+ inclusions (puncta) were counted in E. Data are represented as mean ± SEM. n=5. Significantly different at p=0.0446* in graph E.  VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation were assessed by WB  VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation and p-129-SNCA immunocytochemical analyses. Data are represented as mean ± SEM. n=3. Significantly different at p=0.00003*, 0.0003**, 0.0012***, 0.00004****, 0.0321# in graph G and p=0.0052* in graph H.


Figure 6 Glia derived from mouse VM were transduced with sh - Sgk1 ( B ) or SGK1 ( CMV - Sgk1 , C ) , and co - cultured with VM - derived mDA neurons . Seven days after co - culture , cells were exposed to H2O2 ( 500µM , 3hr ) or not ( - ) , and then mDA neuron viability ( TH + cell counts ) and neurite degeneration ( TH + fiber lengths ) were estimated . Data are represented as mean ± SEM ; n = 4 culture coverslips . Student ' s t - test . Significantly different at p = 0 . 033 * , 0 . 0412 * * , 7 . 44E - 23 # , 1 . 39E - 23 # # in graph B and p = 0 . 0489 * , 0 . 0366 * * , 0 . 0078 # # in graph C . Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner . VM - glia transduced with sh - Sgk1 ( or sh - cont as a control ) were challenged with H2O2 + LPS for 3hr or not . Glial conditioned medium ( GCM ) was prepared in each glial culture condition and administered to mDA neurons primarily cultured from mouse VM ( E ) Cell viability of the neuronal cells was estimated using CCK8 assays and TuJ1 + cell counts . Data are represented as mean ± SEM . n = 3 cultures ; One - way ANOVA . Significantly different at p = 0 . 0002 * , 3 . 29E - 08 * * , 0 . 0003 * * * , 3 . 66E - 06 # , 0 . 0029 # # ( TuJ1 + cell counting ) , 0 . 0127 + , 5 . 98E - 06 + + , 0 . 0002 + + + , 0 . 0005 & , 0 . 0078 & & ( CCK8 assays ) in graph E . F , Effect of the SGK1 inhibitor treatment in the mixed mDA neuron + VM - glia co - cultures . Data are represented as mean ± SEM . n = 3 . Student ' s t - test . Significantly different at p = 0 . 0068 * , 0 . 0368 * * , 0 . 0249 # , 1 . 48E - 27 # # in graph F .

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

Figure 6 Glia derived from mouse VM were transduced with sh-Sgk1 (B) or SGK1 (CMV-Sgk1, C), and co-cultured with VM-derived mDA neurons. Seven days after co-culture, cells were exposed to H2O2 (500µM, 3hr) or not (-), and then mDA neuron viability (TH+ cell counts) and neurite degeneration (TH+ fiber lengths) were estimated. Data are represented as mean ± SEM; n=4 culture coverslips. Student's t-test. Significantly different at p=0.033*, 0.0412**, 7.44E-23#,1.39E-23## in graph B and p=0.0489*, 0.0366**, 0.0078## in graph C.  Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner. VM-glia transduced with sh-Sgk1 (or sh-cont as a control) were challenged with H2O2+LPS for 3hr or not. Glial conditioned medium (GCM) was prepared in each glial culture condition and administered to mDA neurons primarily cultured from mouse VM (E) Cell viability of the neuronal cells was estimated using CCK8 assays and TuJ1+ cell counts. Data are represented as mean ± SEM. n=3 cultures; One-way ANOVA. Significantly different at p=0.0002*, 3.29E-08**, 0.0003***, 3.66E-06#, 0.0029## (TuJ1+ cell counting), 0.0127+, 5.98E-06++, 0.0002+++, 0.0005&, 0.0078&& (CCK8 assays) in graph E.  F, Effect of the SGK1 inhibitor treatment in the mixed mDA neuron + VM-glia co-cultures. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0068*, 0.0368**, 0.0249#, 1.48E-27## in graph F.


Figure 7 ( B - D ) Behaviors of MPTP - PD mice assessed by pole ( B ) , beam ( C ) at 3 - 6 weeks post - MPTP injection , and locomotor activity ( total distance & average speed , D ) tests at 6 weeks post - MPTP injection . n = 20 GSK - 650394 - treated MPTP - PD mice , 14 vehicle - treated MPTP - PD mice , and 8 MPTP - untreated non - PD mice . Data are represented as mean ± SEM . Significantly different at p = 0 . 0012 # , 0 . 0001 * , 3 . 09E - 08 # # , 8 . 21E - 08 * * in graph B and p = 0 . 0009 # , 0 . 0035 * , 1 . 03E - 06 # # , 0 . 00003 * * in graph C and p = 0 . 0001 * , 0 . 0284 * * , 0 . 0101 # ( total distance ) , 1 . 49E - 08 * , 0 . 0005 * * , 0 . 0001 # ( average speed ) in graph D . Histologic assessment of the number of TH + mDA neurons ( E , F ) , neurite lengths ( E , G ) , and cell body sizes ( E , H ) Shown are the representative images for TH + DA neurons in the SN TH + DA neurons in boxed areas of ( E ) are enlarged in right . Histologic assessment of the number of TH + mDA neurons in the SN and TH + fiber intensities in the striatum Shown are the representative images for TH + DA neurons in the TH - immunoreactivity in the striatum , Therapeutic effects of GSK - 650394 in SNCA - PD model mice . Behaviors ( L - N ) of the SNCA - PD mice were assessed at 6 - 12 weeks post - SNCA injection Significantly different at p = 0 . 008 # , 0 . 021 * , 0 . 00001 # # , 0 . 0009 * * in graph L and p = 0 . 00002 # , 0 . 0344 * , 0 . 0005 # # , 0 . 0067 * * in graph M and p = 0 . 0057 # , 0 . 0345 * ( total distance ) , 0 . 0001 # , 0 . 0375 # # , 0 . 0338 * ( average speed ) in graph N . Therapeutic effects of GSK - 650394 in SNCA - PD model mice . mDA neuron degeneration in the SN of the SNCA - PD mice were assessed at 12 weeks post - SNCA injection after sacrifice SNCA pathology assessed by p129 - α - syn immunoreactivity in TH + mDA neurons in the SN . n = 5 GSK - 650394 - treated SNCA - PD mice , 5 vehicle - treated SNCA - PD mice , and 8 SNCA - untreated non - PD mice . Therapeutic effects of GSK - 650394 in SNCA - PD model mice . mDA neuron degeneration in the SN ( O - U ) of the SNCA - PD mice were assessed 12 weeks post - SNCA injection after sacrifice SNCA pathology assessed by p129 - α - syn immunoreactivity in TH + mDA neurons in the SN . n = 5 GSK - 650394 - treated SNCA - PD mice , 5 vehicle - treated SNCA - PD mice , and 8 SNCA - untreated non - PD mice .

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

Figure 7 (B-D) Behaviors of MPTP-PD mice assessed by pole (B), beam (C) at 3-6 weeks post-MPTP injection, and locomotor activity (total distance & average speed, D) tests at 6 weeks post-MPTP injection. n= 20 GSK-650394-treated MPTP-PD mice, 14 vehicle-treated MPTP-PD mice, and 8 MPTP-untreated non-PD mice. Data are represented as mean ± SEM. Significantly different at p=0.0012#, 0.0001*, 3.09E-08##, 8.21E-08** in graph B and p=0.0009#, 0.0035*, 1.03E-06##, 0.00003** in graph C and p=0.0001*, 0.0284**, 0.0101# (total distance), 1.49E-08*, 0.0005**, 0.0001# (average speed) in graph D.  Histologic assessment of the number of TH+ mDA neurons (E, F), neurite lengths (E, G), and cell body sizes (E, H) Shown are the representative images for TH+ DA neurons in the SN TH+ DA neurons in boxed areas of (E) are enlarged in right.  Histologic assessment of the number of TH+ mDA neurons in the SN and TH+ fiber intensities in the striatum Shown are the representative images for TH+ DA neurons in the TH-immunoreactivity in the striatum,  Therapeutic effects of GSK-650394 in SNCA-PD model mice. Behaviors (L-N) of the SNCA-PD mice were assessed at 6-12 weeks post- SNCA injection Significantly different at p=0.008#, 0.021*, 0.00001##, 0.0009** in graph L and p=0.00002#, 0.0344*, 0.0005##, 0.0067** in graph M and p=0.0057#, 0.0345* (total distance), 0.0001#, 0.0375##, 0.0338* (average speed) in graph N.  Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN of the SNCA-PD mice were assessed at 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.  Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN (O-U) of the SNCA-PD mice were assessed 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.


Figure 8 A and B , Efficiency of AAV9 - mediated gene delivery . AA9 viruses expressing the reporter GFP gene were stereotaxically injected into the SN of mice , and GFP expression in astrocytes , microglia , and neuronal cells was quantified . Data are represented as mean ± SEM . n = 3 mice ( > 5 randomly selected microscopic fields per animal were counted ) . Significantly different at p = 0 . 006 * , 5 . 1E - 09 # , 5 . 36E - 09 + in graph B . SGK1 knockdown effect in vivo by treatment with shSgk1 - AAV9 . Total SGK1 mRNA levels were estimated in the midbrain SN of the PD mice injected with shSgk1 ( shControl as control ) AAV9 using real time - PCR ( D ) . Cell type - specific SGK1 knockdown was estimated by counting SGK1 - immuonoreactivity in neuron ( MAP2 + ) , astrocyte ( GFAP + ) , and microglia ( Iba1 + ) populations ( E ) . Data are represented as mean ± SEM . n = 4 mice in each group . Significantly different at p = 0 . 0004 # in graph D and p = 0 . 049 * , 0 . 007 * * , 0 . 002 * * * in graph E . ( F - H ) Behavior of the PD mice assessed by pole ( F ) , beam ( G ) at 6 - 12 weeks , and locomotor activity ( H ) tests at 12 weeks post - SNCA injection . Significantly different at p = 0 . 0003 # , 0 . 0428 * , 7 . 46E - 06 # # , 0 . 0012 * * in graph F and p = 8 . 04E - 08 # , 0 . 004 * , 1 . 62E - 09 # # , 3 . 3E - 06 * * in graph G and p = 0 . 0002 # , 0 . 0194 * ( total distance ) , 9 . 54E - 06 # , 0 . 0047 * ( average speed ) in graph H . Histologic assessment of p129 - SNCA immunoreactivity ( I , J ) , TH + mDA neuron counts ( I , K ) , neurite lengths ( L ) , and cell body sizes ( M ) in the SN Significantly different at p = 0 . 0001 * in graph J , p = 2 . 35E - 25 # , 1 . 09E - 16 # # , 4 . 36E - 08 * in graph K and p = 1 . 5E - 40 # , 1 . 08E - 24 # # , 2 . 32E - 07 * in graph L and p = 9 . 07E - 20 # , 2 . 59E - 16 # # , 0 . 05 * in graph M and p = 4 . 62E - 22 # , 5 . 11E - 12 # # , 3 . 49E - 06 * ( TH ) , n = 7 sh - SGK1 - AAV9 - treated PD mice , 7 sh - cont - AAV9 - treated PD mice , and 8 SNCA - untreated mice . Data are represented as mean ± SEM . One - way ANOVA . Scale bar , 100µm . Histologic assessment of TH + and DA transporter ( DAT ) fiber intensities in the striatum n = 7 sh - SGK1 - AAV9 - treated PD mice , 7 sh - cont - AAV9 - treated PD mice , and 8 SNCA - untreated mice . Data are represented as mean ± SEM . One - way ANOVA . Scale bar , 100µm .

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

Figure 8 A and B, Efficiency of AAV9-mediated gene delivery. AA9 viruses expressing the reporter GFP gene were stereotaxically injected into the SN of mice, and GFP expression in astrocytes, microglia, and neuronal cells was quantified. Data are represented as mean ± SEM. n=3 mice (>5 randomly selected microscopic fields per animal were counted). Significantly different at p=0.006*, 5.1E-09#, 5.36E-09+ in graph B.  SGK1 knockdown effect in vivo by treatment with shSgk1-AAV9. Total SGK1 mRNA levels were estimated in the midbrain SN of the PD mice injected with shSgk1(shControl as control) AAV9 using real time-PCR (D). Cell type-specific SGK1 knockdown was estimated by counting SGK1-immuonoreactivity in neuron (MAP2+), astrocyte (GFAP+), and microglia (Iba1+) populations (E).  Data are represented as mean ± SEM. n=4 mice in each group. Significantly different at p=0.0004# in graph D and p=0.049*, 0.007**, 0.002*** in graph E. (F-H) Behavior of the PD mice assessed by pole (F), beam (G) at 6-12 weeks, and locomotor activity (H) tests at 12 weeks post- SNCA injection. Significantly different at p=0.0003#, 0.0428*, 7.46E-06##, 0.0012** in graph F and p=8.04E-08#, 0.004*, 1.62E-09##, 3.3E-06** in graph G and p=0.0002#, 0.0194* (total distance), 9.54E-06#, 0.0047* (average speed) in graph H.  Histologic assessment of p129-SNCA immunoreactivity ( I,J), TH+ mDA neuron counts (I,K), neurite lengths (L), and cell body sizes (M) in the SN Significantly different at p=0.0001* in graph J, p=2.35E-25#, 1.09E-16##, 4.36E-08* in graph K and p=1.5E-40#, 1.08E-24##, 2.32E-07* in graph L and p=9.07E-20#, 2.59E-16##, 0.05* in graph M and p=4.62E-22#, 5.11E-12##, 3.49E-06* (TH), n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm.  Histologic assessment of TH+ and DA transporter (DAT) fiber intensities in the striatum n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm.


Figure 9 A - C , Quantitative and morphometric analyses of microglia in the SN area of SNCA - PD mice . Shown in A are representative views of Iba1 + microglia neighboring TH + mDA neurons in the SNs of PD mice treated with GSK - 650394 and vehicle . Insets , enlarged views for the boxed areas . Data are represented as mean ± SEM . n = 3 mice per group . Significantly different at p = 1 . 49E - 11 * in graph B and p = 0 . 000003 * in graph C . D and E , The M1 marker CD16 / 32 expression in Iba1 + microglia . Data are represented as mean ± SEM . n = 3 mice per group . Significantly different at p = 0 . 0016 * in graph E . F and G , mRNA expression of NLRP3 - inflammasome components and pro - inflammatory cytokines in MPTP ( F ) and SNCA ( G ) PD mice injected with the SGK1 inhibitor or vehicle ( control ) . Each bar represents the mean ± SEM of 3 PCR values from each animal , n = 3 mice per group . Significantly different at p = 0 . 0372 * , 0 . 0173 * * , 0 . 0003 * * * in graph F and p = 0 . 002 # , 0 . 0113 # # , 0 . 0053 # # # , 0 . 0001 # # # # in graph G . H - J , ASC expression in the SNs and SN microglia of SNCA - PD mice . n = 5 mice per group . Data are represented as mean ± SEM . Significantly different at p = 1 . 91E - 09 * in graph I and p = 0 . 0079 * in graph J . K and L , β - gal - stained cell senescence in SNCA - PD mouse SN . Data are represented as mean ± SEM . n = 5 mice per group . Significantly different at p = 0 . 0001 *

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Figure 9 A-C, Quantitative and morphometric analyses of microglia in the SN area of SNCA-PD mice. Shown in A are representative views of Iba1+ microglia neighboring TH+ mDA neurons in the SNs of PD mice treated with GSK-650394 and vehicle. Insets, enlarged views for the boxed areas. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=1.49E-11* in graph B and p=0.000003* in graph C.  D and E, The M1 marker CD16/32 expression in Iba1+ microglia. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=0.0016* in graph E.  F and G, mRNA expression of NLRP3-inflammasome components and pro-inflammatory cytokines in MPTP (F) and SNCA (G) PD mice injected with the SGK1 inhibitor or vehicle (control). Each bar represents the mean ± SEM of 3 PCR values from each animal, n=3 mice per group. Significantly different at p=0.0372*, 0.0173**, 0.0003*** in graph F and p=0.002#, 0.0113##, 0.0053###, 0.0001#### in graph G.  H-J, ASC expression in the SNs and SN microglia of SNCA-PD mice. n=5 mice per group. Data are represented as mean ± SEM. Significantly different at p=1.91E-09* in graph I and p=0.0079* in graph J.  K and L, β-gal-stained cell senescence in SNCA-PD mouse SN. Data are represented as mean ± SEM. n=5 mice per group. Significantly different at p=0.0001*


Figure 1CD14 + monocytes were cultured with M - CSF , IL - 4 and TNFα and analyzed by scRNA - seq at different time points ( n = 2 biological replicates ) . ( D ) Expression patterns of monocytes ( top row ) , macrophages ( middle row ) and DC ( bottom row ) markers .

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Figure 1CD14+ monocytes were cultured with M-CSF, IL-4 and TNFα and analyzed by scRNA-seq at different time points (n=2 biological replicates). (D) Expression patterns of monocytes (top row), macrophages (middle row) and DC (bottom row) markers.


Figure 2 ( D ) Expression of phenotypic markers used for UMAP projection . ( E ) Supervised gating strategy for moDC and moMac at each time point ( gated on live CD11b + CD115 + cells ) . ( F ) Supervised gating strategy to identify monocyte - derived cells .

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Figure 2(D) Expression of phenotypic markers used for UMAP projection.(E) Supervised gating strategy for moDC and moMac at each time point (gated on live CD11b+ CD115+ cells).(F) Supervised gating strategy to identify monocyte-derived cells.


Figure 3 ( E ) Transcription factor activity was calculated with Dorothea and Viper . Differentially activated regulons between clusters are shown .

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Figure 3(E) Transcription factor activity was calculated with Dorothea and Viper. Differentially activated regulons between clusters are shown.