Additional file 1 of The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation

Additional file 1: Supplementary Figures 1-6.SupplementaryFig. 1. a. Function clustering of DEGs. b-c. Huh7 (b) and HepG2 (c) cells were treated with GANT61 (20 μM) for 48 h and harvested for real-time PCR analysis with the indicated primers. d-e. Previous Hh target screening via microarray. f-g. LM-3 cells were cells were treated with GANT61 (f) and Cyclopamine (g) for 48 h and harvested for real-time PCR analysis with the indicated primers. Tomatidine was used as control for Cyclopamine. h-i. Huh7 (h) and HepG2 (i) cells were treated with Cyclopamine (20 μM) for 48 h and harvested for real-time PCR analysis with the indicated primers. Tomatidine was used as control. Data was shown as mean ± SD (n = 3). *, p < 0.05; **, p < 0.01. Supplementary Fig. 2. a. Validation of SK-Hep-1 cells stably over-expressing TPX2 using WB analysis with the indicated antibodies. b. Comparison of the proliferative ability of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in SK-Hep-1 cells treated with EdU. Scale bar, 100 μm. c. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). d. Cell growth curves of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in SK-Hep-1 cells. e-f. Comparison of the proliferative ability of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in HepG2 cells using colony formation assay. Colonies were counted using the ImageJ software. Data was shown as mean ± SD (n = 3). *, p < 0.05; **, p < 0.01, N.S. denotes not significant. Supplementary Fig. 3. a. Validation of Hep3B sh-TPX2 stable cell lines using WB analysis. b. TPX2 abrogation inhibited Hh signaling-induced HCC cell proliferation as determined using EdU staining. Scale bar, 100 μm. c. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). d. Cell growth curves of sh-Control / Shh (−), sh-Control / Shh (+), sh-TPX2 / Shh (−), and sh-TPX2 / Shh (+) in Hep3B cells. e-f. Comparison of the proliferative ability of sh-Control / Shh(−), sh-Control / Shh (+), sh-TPX2 / Shh(−), and sh-TPX2 / Shh (+) in Hep3B cells using colony formation assay. Colonies were counted using the ImageJ software. g. Marker of cell proliferation, PCNA, were determined via WB analysis. Data was shown as mean ± SD (n = 3). *, p < 0.05; **, p < 0.01. Supplementary Fig. 4. a. Validation of Huh7 sh-TPX2 stable cell lines using quantitative real-time PCR. b. TPX2 abrogation inhibited Hh signaling-induced HCC cell proliferation as determined using EdU staining. Scale bar, 100 μm. c. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). Supplementary Fig. 5. a-d. The expression of FOXM1 in Fig. 4d (a), 4e (b), 4f (c) and 4 g (d) were testified via immunoblotting. Supplementary Fig. 6. a. Real-time PCR to determine the mRNA levels of TPX2 and FOXM1 in Huh7 and Hep3B cells with stable knockdown of FOXM1. b. Real-time PCR analysis to determine the mRNA levels of TPX2 and FOXM1 in Huh7 and HepG2 cells stably over-expressing FOXM1. Supplementary Fig. 7. a. Expression of FOXM1 and TPX2 in HCC tissue and normal tissue based on TCGA. b. Expression of FOXM1 and TPX2 in HCC based on tumor grade in UALCAN database. **, p<0.01. c. Correlation analysis of TPX2 and FOXM1 mRNA levels in TCGA database (n = 381). d-e. Five human HCC cell lines showed varying protein (d) and mRNA (e) expression of FOXM1 and TPX2. f-g. Kaplan-Meier overall survival rate analysis of a set of HCC cancer patients grouped by FOXM1 (f) and TPX2 (g) levels in TCGA dataset. h-i. Kaplan-Meier progression-free survival rate analysis of a set of HCC cancer patients grouped by FOXM1 (h) and TPX2 (i) levels in the Kaplan-Meier Plotter website.