Crosstalk between heat shock factor 1 and signal transducer and activator of transcription 3 mediated by interleukin-8 autocrine signaling maintains the cancer stem cell phenotype in liver cancer

Table S1. The list of real-time PCR primers used in this study.

Table S2. Correlation between HSF1^HighSTAT3^Low and HSF1^HighSTAT3^High expression and clinicopathologic characteristics of liver cancer patients

Table S3. Correlation between HSF1^LowSTAT3^High and HSF1^HighSTAT3^High expression and clinicopathologic characteristics of liver cancer patients

Table S4. Correlation between HSF1^LowSTAT3^Low and HSF1^HighSTAT3^High expression and clinicopathologic characteristics of liver cancer patients

Figure S1. The expression of CSC markers and signaling in HepG2 and HepG2-R xenograft tumors was examined by immunohistochemistry with antibodies (Abs) against P-gp, CD24, CD133, β-catenin, p-HSF1, and p-STAT3. Bar = 200 μm.

Figure S2. The integral density of the protein bands of p-HSF1, HSF1 and GAPDH shown in Fig. 2A (left) and Fig. 2G (right) was analyzed with ImageJ software. The experiments were repeated two times. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure S3. A, HepG2 and HepG2-R cells were cotransfected with pGL3.basic-HSF1 promoter and pGMLR-TK vectors and cultured for 60 h. HSF1 transcriptional activity was analyzed by luciferase assay. B, HSF1 mRNA levels in HepG2 and HepG2-R cells were detected by real-time RT–PCR. C, HepG2 cells were cotransfected with the pGL3.0 basic-ABCB1 promoter and pGMLR-TK vectors and cultured for 60 h. Then, the cells were heated for 1 h at 43°C and allowed to recover for 2 h at 37°C. Unheated cells were used as controls. ABCB1 transcriptional activity was analyzed by luciferase assay. *p<0.05; **p<0.01; ***p<0.001.

Figure S4. Effect of HSF1 knockdown on HSP27 expression. HepG2-R cells were transfected with scramble or HSF1-shRNA plasmids and treated with or without 31.25 nM adriamycin for 48 h. The expression of HSP27 and GAPDH was analyzed by Western blotting.

Figure S5. Knocking down HSF1 expression led to upregulated IL-6 mRNA expression and STAT3 activation in HCC cells. HepG2-R and HuH-7-R cells were transfected with pLKO.1-scramble and HSF1-shRNA vectors and cultured for 60 h. IL-6 mRNA levels were detected by real-time RT–PCR (A and B). The experiments were repeated three times. ***p<0.001; ****p<0.0001. HepG2 cells were starved for 2 h in serum-free medium and then treated with rhIL-6 (10 ng/ml) for the indicated durations. The expression of p-STAT3, STAT3, p-HSF1, HSF1, and GAPDH was detected by Western blotting (C).

Figure S6. Effect of exogenous overexpression of HSF1 on STAT3 activation. HepG2-R cells were transfected with pCDH-MCS-CMV-EF1-puro and HSF1-OX vectors and cultured for 72 h. The expression of p-HSF1 (Ser326), HSF1, p-STAT3 (Y705), STAT3, and GAPDH was analyzed by Western blotting.

Figure S7. The proliferation inhibitory effects of KRIBB11 and Stattic in combination with adriamycin. HepG2-R cells were seeded in 96-well plates in triplicate at an initial cell density of 3×103 cells/well. KRIBB11 (2.5 μM, left) and Stattic (2.5 μM, right) in combination with or without 500 nM adriamycin. The in vitro proliferation activity was analyzed by MTS assay. The experiments were repeated two times. ***p<0.001; ****p<0.0001.

Figure S8. A, The expression of p-HSF1, HSF1, p-STAT3, STAT3, p-ERK, ERK, p-Akt, Akt, and GAPDH in WRL-68, LO2, HepG2, SMMC-7721, HuH-7, and SMMC-7402 cells was analyzed. B, P-HSF1, HSF1, p-STAT3, and STAT3 bands of HepG2, HepG2-R, SMMC-7721, HuH-7, HuH-7-R, and SMMC-7402 cells were quantified with ImageJ software. The intensities represents the ratio of p-HSF1 to HSF1 and p-STAT3 to STAT3. The Pearson correlation between the intensity of p-HSF1 and p-STAT3 was analyzed by GraphPad Prism 6.0.

Figure S9. A, The mRNA levels of IL-8 in the relapsed and nonrelapsed HCC patient samples in the TCGA database were analyzed by GraphPad Prism 6.0. B, The correlation between CXCL-8 expression and overall survival of liver cancer patients was predicted by GEPIA (http://gepia.cancer-pku.cn/).

Figure S10. Upregulation and activation of HSF1 directly regulated the transcriptional activity of IL-8 in HCC cells. HepG2-vector and HepG2-HSF1-OX cells were transfected with pGL4.20-IL-8 promoter and pRL-TK vectors and cultured for 48 h. Then, the cells were treated with various concentrations of adriamycin (0, 62.5, and 125 nM) for 24 h. IL-8 transcriptional activity was analyzed by luciferase assay. The experiments were repeated three times. **p<0.01; ***p<0.001; ****p<0.0001.

Figure S11. IL-8 mediates the coactivation of HSF1-STAT3 and the expression of CSC markers in HCC cells. HepG2 cells were cultured overnight and starved for 2 h in serum-free medium and then treated with rhIL-8 (50 ng/ml) for the indicated durations. The expression of p-STAT3, STAT3, p-HSF1, HSF1, P-gp, CD133, CD24, β-catenin, and GAPDH was detected by Western blotting.

Figure S12. IL-8 increased the transcriptional activity of HSF1 and STAT3 reporters in HCC cells. HepG2 cells were transfected with a pGL4.26-HSF1-reporter (A) and pGL4.26-STAT3-reporter (B). After 48 h, the transfected cells were treated with 20 ng/ml rhIL-8 for 0, 6, and 9 h. The transcriptional activity of HSF1 and STAT3 was analyzed by luciferase assay. The experiments were repeated three times. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure S13. IL-8 neutralization antibodies reversed the CSC phenotype acquired by liver cancer cells. HepG2-R cells were treated with 0, 50, and 100 ng/ml neutralizing antibody against human IL-8 for 24 h. The expression of CD133, OCT-4, β-catenin, and GAPDH was assessed by Western blotting.

Figure S14. HuH-R cells were cultured in medium with or without rhIL-8 (50 ng/ml) for 48 h. The IL-6, IL-8, and TNF-α mRNA levels were determined by real-time PCR. ****p<0.0001.

Figure S15. Effect of knocking down HSF1 and STAT3 expression on IL-8 autocrine signaling. HepG2-R and HuH-R cells were seeded in 60-mm dishes, and then HSF1 and STAT3 expression was knocked down independently or in combination by lentiviral shRNA vectors and cultured for 72 h. The secretion of IL-8 in the medium was detected by ELISA. The experiments were repeated three times. **p<0.01; ***p<0.001.

Figure S16. Antitumor effect of targeting HSF1 and STAT3 alone or in combination in mice bearing liver cancer xenografts. HepG2-R cells (5×106) were injected subcutaneously into the right upper flanks of the mice. Seven days after the implantation of tumor cells, the mice were assigned to four groups and treated with vehicle, 30 mg/kg KBRBB11, 15 mg/kg Stattic, or 30 mg/kg KBRBB11+15 mg/kg Stattic daily by intraperitoneal (i.p.) injection for nearly 2 wk. All mice were sacrificed. The primary tumors were dissected, photographed (A), and weighed (B). C, Volume of xenograft tumors following treatments. The experiments were repeated three times. *p<0.05; **p<0.01.