Fig 2: Signaling pathways in KC and qKC mice. (A) Representative immunohistochemical staining images for Ki67, PCNA, cyclinD1, p-ERK, and COX-2 in the pancreases of control, qKO, KC, and qKC mice at 3 months of age, and 9 month old KC and qKC mice. Strong nuclear staining of Ki67 detected in PanIN lesions of KC and qKC mice, and acinar cells of control, qKO, KC, and qKC animals (black arrowheads). No Ki67 expression in islets of control mice (red arrow). Positive staining of PCNA in acinar cells, islets, and ducts in all experimental animals (black arrowheads). Absence of cyclin D1 immunoreactivity in control and qKO mice (red arrows). Strong nuclear labeling of cyclin D1 in all KC and qKC pancreases (black arrowheads). P-ERK expression visible in islets of control, qKO, KC, and qKC animals (yellow arrows). In addition, strong positive staining of p-ERK noted in PanINs (black arrowheads) and stroma of KC and qKC mice (black asterisks). No expression of p-ERK in acinar cells of all experimental animals (red arrows). Cytoplasmic labeling of COX-2 detected only in PanIN lesions occurring in KC and qKC mice (black arrow). Absence of COX-2 immunoreactivity in control and qKO pancreases. Scale bars represent 100 µm. (B) Histological score of Ki67-, PCNA-, cyclin D1-, p-ERK-, and COX-2-positive cells in pancreas of experimental mice groups (control mice, n = 10; qKO mice, n = 10; KC mice of age 3 months, n = 14, 9 months, n = 5; qKC mice of age 3 months, n = 9, 9 months, n = 8). Error bars represent mean ± SD; Error bars represent mean ± SD; * p < 0.05, ** p < 0.01, *** p = 0.0001, **** p < 0.0001; ns = not significant (Student’s t-test).

Fig 3: Effect of BC-1215 on NF-?B and I?B-a activation in lung tissues. BC-1215 reduced nuclear NF-?B p65 and cytoplasmic phospho-NF-?B p65 levels (A) and increased I?B-a levels (B) in ischemia-reperfusion (I/R)-induced lung injury. PCNA and ß-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. Representative blots are shown. Data are expressed as mean ± SD (n = 3 per group). *** p < 0.001, compared with the control group;++p < 0.01, +++p < 0.001 compared with the I/R group.

Fig 4: The effect of IRF9 on neointima formation is mediated by SIRT1.(a,c) HE-stained sections show the structures of carotid arteries from WT, SMC-SIRT1-KO, IRF9-KO and DKO mice (a) or NTG, SMC-SIRT1-TG, SMC-IRF9-TG and DTG mice (c) at 28 days post injury. The insets in the upper panels were magnified, and the magnified images are presented in the lower panels. The black arrows indicate inner elastic discs. Scale bar, 50 µm. The intimal areas and intima/media ratios were quantified (a, n=5–12 per group, c, n=6–10 per group). (b,d) Immunofluorescence staining shows PCNA, Cyclin D1 (red), a-SMA and SM22a (green) expression and localization in carotid arteries of WT, SMC-SIRT1-KO, IRF9-KO and DKO mice (b) or NTG, SMC-SIRT1-TG, SMC-IRF9-TG and DTG mice (d) at 28 days post injury. DAPI (blue) staining indicates the nuclei. Scale bar, 20 µm. The proportion of PCNA-positive cells and OD values for Cyclin D1, a-SMA and SM22a were calculated ((b) n=3–5 per group, (d) n=3–6 per group). (a–d) The values are presented as the mean±s.d., *P<0.05 versus the WT or NTG group, #P<0.05 versus the SMC-SIRT1-KO group or the SMC-SIRT1-TG group, †P<0.05 versus the IRF9-KO group or the SMC-IRF9-TG group. Statistical analysis was carried out using one-way ANOVA test.

Fig 5: AKT inhibition impairs PCNA ubiquitylation. a U2OS cells were UV irradiated (15 J/m2) and treated for 12 h with the indicated inhibitors at 1 µM. The western blot shows the strong PCNA ubiquitylation inhibitory activity found in two structurally related hits: C11 (compound #: GSK1581428A) and G8 (compound #: GSK1389063A). The graph in the lower panel shows the quantification of three independent experiments. Statistical analysis was performed using analysis of variance (ANOVA) with Tukey Kramer post-test (***p = 0.001). b U2OS cells were treated as in a and western blots with specific antibodies were performed to study pAKT, total AKT, and p-GSK3ß levels. a-Tubulin was used as a loading control. c U2OS cells were pre-treated for 12 h using 0.5 µM C11 and 5 µM of the structurally unrelated AKT inhibitors: MK-2206 (Merck), AZD5363 (AstraZeneca), GSK690693 (GlaxoSmithKline). After UV, all these inhibitors were used at 20 µM and C11 was used at 1 µM. The normalized ubi-PCNA/total PCNA ratios are shown below the PCNA panel. pAKT, AKT, p-GSK3ß, and p-PRAS40 western blots were performed at 3 and 12 h post-treatment to confirm the AKT inhibitory activity of each compound. d U2OS cell were transfected with two concentrations of siRNAs. Forty-eight hours later, cells were UV irradiated, and after 12 h, samples were processed for quantification of PCNA ubiquitylation by western blot. A western blot for pan-AKT was performed to confirm the siRNA-mediated knockdown. The normalized ubi-PCNA/total PCNA ratios are shown below the PCNA panel