Fig 2: Sirt3 deficiency triggered LPC induced ß cell dedifferentiation. A: Relative mRNA expression levels of Sirt3 and Atx were assessed by RT-PCR in MIN6 cells after knockdown of Sirt3; B, C: Relative mRNA expression levels of ß cell identity genes, Ins2, progenitor cell markers and a cell enriched genes were measured by RT-PCR in normal and Sirt3 deficiency MIN6 cells after treatment with 0.1 µmol/L LPA or LPC. D: Representative western immunoblot data (total protein) of SIRT3, ATX, ß cell identity markers, progenitor cell markers and a cell enriched markers in normal and Sirt3 deficiency MIN6 cells with 0.1 µmol/L LPC treatment for 24 h (n = 3 biological replicates). E-G: Immunocytochemistry of NKX6.1 (E), PDX1 (F) and ALDH1A3 (G) in primary islets isolated from Sirt3f/f and Sirt3f/f;Cre/+ mice with 0.1 µmol/L LPC or LPA treatment for 24 h (scale bar, 25 µm). Green, Insulin; red, NKX6.1 (E), PDX1 (F) and ALDH1A3 (G); blue, Hoechst. H-J: Quantification of relative fluorescence positive areas shown in E-G (n = 6–8 islets). Data are presented as Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by One-way ANOVA followed by Bonferroni post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: RNF8 depletion inhibits cell proliferation and enhances enzalutamide sensitivity in prostate cancer cell lines.A, B Control and stable RNF8 knockdown LNCaP (A) and 22Rv1 (B) cells were treated with vehicle or DHT (10-8 M) for 10 days. The cell colonies were stained with Coomassie blue. C, D Control and stable RNF8 knockdown LNCaP (C) and 22Rv1 (D) cells were treated with vehicle or DHT (10-8 M) for 0, 1, 2, 3 days. The cell viability was detected by the CCK8 proliferation assay. Data were means ± SEM (n = 3). **P < 0.01; ***P < 0.001; **** P < 0.0001 (two-sided unpaired t-test). E The proliferation of control and stable RNF8 knockdown DU145 cells was examined by the CCK8 assay. Data were means ± SEM (n = 3). ns no significance (two-sided unpaired t-test). RNF8 knockdown efficacy was determined by western blot. Numbers under the western blot bands indicate the relative gray value normalized to ß-actin. F The photograph for the xenografts from NOD/SCID mice injected subcutaneously with control and stable RNF8 knockdown 22Rv1 cells (n = 8). G, H The median of tumor volume (G) and tumor weight (H) from (F). **P < 0.01 (two-sided Mann–Whitney test). I Representative images of xenograft tumor tissues in (F) staining with RNF8, FASN, and ALDH1A3. The H&E staining was also presented. Scale bar, 50 µm. J Statistical analysis of the mean staining intensity of RNF8, FASN, and ALDH1A3 in xenograft tumor tissues in (I). **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-sided unpaired t-test). K Control and stable RNF8 knockdown 22Rv1 cells were treated with vehicle or enzalutamide (10 µM) for 10 days. The cell colonies were stained with Coomassie blue. L, M Control and stable RNF8 knockdown 22Rv1 (L) and LNCaP-EnzR (M) cells were treated with vehicle or enzalutamide (10 µM) for 0, 1, 2, 3 days. The cell viability was detected by the CCK8 assay. Data were means ± SEM (n = 3). *P < 0.05; ***P < 0.001; ****P < 0.0001 (two-sided unpaired t-test).

Fig 4: Specific knockdown ATX improved HFD-induced ß cell dedifferentiation in Sirt3f/f;Cre/+ islets in mice. Sirt3f/f and Sirt3f/f;Cre/+ mice were injected with AAV-Atx-shRNA (for specific knocking down ATX in pancreatic ß cells) or AAV-scramble respectively, followed by feeding with HFD for 6 weeks before sacrifice. A-F: Immunofluorescent staining of insulin and JNK/p38 MAPK pathway or ß cell dedifferentiation markers in mouse pancreas (scale bar, 50 µm). Green, p-JNK (A), p-p38 MAPK (B), NKX6.1 (C), PDX1 (D), ALDH1A3 (E) and NGN3 (F); red, insulin; blue, DAPI. G: GSIS was measured in isolated primary islets from four groups of mice (n = 3 mice per group). H, I: Relative mRNA expression levels of ß cell identity genes (including Foxo1, Nkx6.1 and Pdx1), Ins2, progenitor cell markers (Aldh1a3, Ngn3, Sox9 and Oct4) and a cell enriched genes (such as Arx and Mafb) were assessed by RT-PCR in isolated primary islets from four groups of mice (n = 3 mice per group). Data are presented as Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by One-way ANOVA followed by Bonferroni post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)