Fig 1: Smad6 promotes STAT3 activation through inhibition of PIAS3. a, b Nuclear-Smad6 regulated STAT3 downstream genes expression in GBM cells. The expression of proteins downstream of STAT3 signaling (c-jun, CCND1, and Sox2) were analyzed by western blot in glioma cells with Smad6 KD (a) or nuclear-Smad6 OE (b). The quantification of indicated proteins was listed. c Nuclear-Smad6 enhanced the enrichment of STAT3 on the promoter of STAT3 downstream targets. The enrichment of STAT3 binding to the promoter of target genes (SOX2 and CCND1) was examined by ChIP-qPCR in glioma cells with nuclear-Smad6 OE (left panel) or KD (right panel) (n = 3). d Smad6 KD or e nuclear-Smad6 OE did not influence the phosphorylation (Y705) of STAT3, but regulated PIAS3 expression. f Nuclear-Smad6 rescued STAT3 transcriptional activity that was inhibited by PIAS3 (n = 3). Luciferase assay of STAT3 transcriptional activity in HEK293 cells transfected with HA-PIAS3 and/or indicated doses (μg) of Flag-Smad6-NLS. IL-6 (25 ng ml−1) was added 6 h before the assay. Data were represented as means ± SD and analyzed using two-tailed Student’s t-test. **P < 0.01
Fig 2: MH2 domain is essential for Smad6-mediated PIAS3 ubiquitination and proteasomal degradation. a Smad6-MH2 deletion mutant failed to induce PIAS3 ubiquitination and degradation. 293T cells were transfected with expression constructs in the indicated combinations. b Nuclear-MH2 antagonized the ubiquitination and degradation of PIAS3 induced by nuclear-Smad6 in a dose-dependent manner. 293T cells were transfected with expression constructs in the indicated combinations and concentrations (0, 0.5, 1.0 μg per well in a 6-well plate). c Nuclear-MH2 rescued nuclear-Smad6 induced PIAS3 downregulation in A172 and T98G cells. d Nuclear-MH2 increased endogenous PIAS3 levels in U87 and U251 cells. e Nuclear-MH2 inhibited tumor growth in nude mice. U87 cells with nuclear-MH2 OE or Mock were used for tumor formation in a nude mouse xenograft model, and mean tumor volumes and average tumor weight of xenograft tumors were measured (n = 6). f MH2 introduction increased PIAS3 expression and impaired STAT3 downstream genes (CCND1 and SOX2) expression in xenograft tumors (n = 6). Data were represented as means ± SD and analyzed using two-tailed Student’s t-test. **P < 0.01
Fig 3: Integrator together with NACK activates Notch-mediated transcription. a MS/MS analysis shows that subunits of the Integrator complex co-purify with the Notch Ternary Complex in the Notch-driven T-cell lymphoma cell line 4084 by affinity chromatography. Notch-independent lymphoma 6780 was used as a negative control. b Representative ChIP on the HES1, HES4, and CCND1 promoters in OE19 EAC. Bars represent standard deviation (N = 3). c Expression of Notch1 target genes in OE19 and OE33 upon INTS11 knockdown. Bars represent standard deviation (the mean from 3 biological replicates). **p < 0.01 versus CTR sample. d Expression of Notch1 target genes in OE19 and OE33 upon NACK knockdown. Bars represent standard deviation (the mean from 3 biological replicates). **p < 0.01 versus CTR sample
Fig 4: STAT3 KD attenuates histone modification and RNA Pol II recruitment on target gene promoters. MB cells were treated with 0.5 μg/mL Dox prior to crosslinking with formaldehyde. STAT3, BRD4, pSer2-RNAPol II, and H3K27Ac binding to the MYC (A) and CCND1 (B) promoters were demonstrated by ChIP-qPCR. IgG Ab was used as a negative control and data are presented as percent (%) input; ** p < 0.01, *** p < 0.001. (C) Cells were treated the same as before; binding of STAT3 and p300 on the MYC promoter are presented as fold change compared to IgG; ** p < 0.01, *** p < 0.001. (D) HD-shSTAT3 and ONS-shSTAT3 MB cells were treated either with or without Dox and WCEs were analyzed for H3K27Ac expression. Total H3 and GAPDH were used as control. (E) Cells were grown in coverslips and were treated as in (D) and an immunofluorescence (IF) experiment was performed to detect the levels of H3K27Ac. Nuclear staining is shown by DAPI. The uncropped bolts are shown in Supplementary Materials file S1.
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