Fig 1: 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.
Fig 2: Effect of STAT3 genetic KD on STAT3 targets in vitro. (A) STAT3 mRNA expression in HD-shSTAT3 and ONS-shSTAT3 MB cells expressing Dox-inducible STAT3 shRNA was validated by qPCR. Cells were either treated with Dox (0.5 μg/mL) for 48 h or left untreated before harvesting the RNA. ** Represents p < 0.01 for Dox-treated cells versus control cells. (B) HD-shSTAT3 and ONS-shSTAT3 MB cells were treated as in (A) prior to making the whole cell extract (WCE). Expression levels of STAT3, pY705-STAT3 and pS727-STAT3 were analyzed by Western immunoblot analysis and GAPDH was used as a loading control. (C) Cells were treated as in (A) and STAT3 target gene expression was analyzed by qPCR. * Represents p < 0.05, ** p < 0.01. (D) WCEs from Dox-treated HD-shSTAT3 and ONS-shSTAT3 MB cells were analyzed by Western blot for the expression levels of STAT3 target genes. Cyclophilin B acts as a loading control. (E) Enrichment of MYC target genes is shown in HD-shSTAT3 and ONS-shSTAT3 cells after STAT3KD by GSEA plot. FDR represents false discovery rate; FDR q < 0.25 is considered significant. The uncropped bolts are shown in Supplementary Materials file S1.
Fig 3: STAT3 KD confers chemosensitivity to MB cells and tumors. (A) HD-shSTAT3 and (B) ONS-shSTAT3 cells were treated either with increasing concentrations of cisplatin alone or in combination with 0.5 μg/mL Dox for 48 h and cell viability was measured by MTT assay; * p < 0.05, ** p < 0.01. (C) HD-shSTAT3 cells were treated with either Dox or cisplatin alone or in combination or left untreated for 12 days. Colonies formed were fixed, stained (left) and counted from triplicate wells and are shown in a bar diagram (right); ** p < 0.01 (D) STAT3 target gene expressions were analyzed in HD-shSTAT3 and (E) ONS-shSTAT3 cells by qPCR, either in the presence of Dox or cisplatin alone, or in combination; * p < 0.05. (F) HD-shSTAT3 and (G) ONS-shSTAT3 cells (0.5 × 106) were subcutaneously implanted in 5- to 6-week-old athymic nude mice. Mice were given water supplemented either with Dox (2 mg/mL in drinking water) or saline (vehicle), which was continued throughout the experiment. Cisplatin (2 mg/kg) was injected twice a week intraperitoneally or in combination with Dox. The tumor size in treated groups (Dox, cisplatin and Dox + cisplatin) was compared with that of vehicle. Tumor volume was measured at indicated days and a tumor growth curve was plotted (top). Resected xenograft tumors (bottom) after completion of treatment were shown. Plotted values and error bars represent mean ± SEM. (H) Representative IHC images (20×) of the xenograft sections of Ki-67, caspase-3, MYC and pSTAT3 are shown. The percentage of Ki-67-, caspase-3-, MYC- and pSTAT3-positive cells derived from histology scores were quantified in the three tumor sections of each treatment group; *** p < 0.001 (ANOVA).
Fig 4: WP1066 attenuates MB tumor growth, augments chemosensitivity and mice survival. (A) HD-MB03 cells (0.5 × 106) were subcutaneously implanted in 5- to 6-week-old athymic nude mice. When palpable tumors were reached, treatment was initiated. WP1066 (30 mg/kg) was given 3 days a week for 4 weeks and cisplatin (2 mg/kg) twice a week for 4 weeks. DMSO (5%) was given as vehicle control. Tumor volume was measured at indicated days and tumor growth curves (Vehicle, WP1066, cisplatin and WP1066 + cisplatin) were plotted. (B) Survival curves were plotted using a Kaplan–Meier analysis. The p value was calculated using a 2-sided log-rank test. (C) Representative IHC images (20×) of the xenograft sections of Ki-67, caspase-3, MYC and pSTAT3 are shown. The percentages of Ki-67-, caspase-3-, MYC- and pSTAT3-positive cells derived from histology scores were quantified in the three tumor sections of each treatment group and are shown in a bar diagram. (D) Luciferase-expressing HD-MB03 cells were implanted orthotopically into the cerebella of NSG mice for tumor growth and the mice were treated with either WP1066 or cisplatin or the combination of both. IVIS images of representative mouse from each treatment group at day 5 (treatment started), and at day 21 (mice sacrificed) after implantation are shown. (E) H&E staining and IHC staining of cerebellar section of mice brain show the presence of tumor and proliferating cells; ** p < 0.01, *** p < 0.001 (ANOVA). (F) Proposed working model by which activated STAT3 and its co-regulators promote oncogene expression and chemoresistance in MB and how it could be targeted by STAT3 inhibition.
from Cell Signaling Technology for SimpleChIP ® Human c-Myc Intron 1 Primers