Fig 2: CARMA3 regulated the expression of STMN1 via the NF-?B pathway. A, Reverse-transcription polymerase chain reaction (RT-PCR) analysis of the STMN1 expression level in CARMA3-shRNA-knockdown clear cell renal cell carcinoma (ccRCC) cells. B, Western blot analysis of the I?Ba phosphorylation level in CARMA3-shRNA-knockdown ccRCC cells. C, RT-PCR analysis of the STMN1 expression level after application of the pyrrolidinedithiocarbamic acid NF-?B inhibitor. D, Luciferase assays of the NF-?B1 transcription activity between the wild-type and mutated STMN1 promotors. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 3: FoxM1-mediated cancer cell proliferation requires STMN1 expression. a The hepatocellular carcinoma cell line SMMC-7721, gastric cancer cell line SGC-7901, colorectal cancer cell lines HCT 116 and HT-29 were used to established FoxM1-silenced and STMN1-overexpressed cell lines by lentivirus-mediated system. The protein levels of FoxM1 and STMN1 were detected by Western blot and the mRNA levels were detected by RT-qPCR. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. b Cell viability of cells was detected by cell counting-8 kit. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. c Plate clone assay was performed and the number of clones was measured by ImageJ software. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. d Cell cycle was analyzed by flow cytometry. e Cell mitosis was analyzed by immunofluorescence of a-Tubulin. The nuclei (blue) are stained with DAPI and the a-Tubulin (red) is stained with Alexa Flour 555. The images were captured at 60× magnification, and the scale bar is 10 µm. The yellow arrow points to the normal mitosis cells. The percentage of normal or abnormal cells was calculated by numbers of normal or abnormal nucleus divided by numbers of total nucleus × 100%. The data were presented as the mean ± SD of three different fields of view at low magnification (20×). The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01. f Colorectal cancer cell lines HTC 116 were infected with lentivirus. The mice were separated into four groups, control group (cells infected with pLKO.1-control lentivirus and pLV-GFP lentivirus), FoxM1-knockdown group (cells infected with pLKO.1-shFoxM1 lentivirus and pLV-STMN1-GFPSpark lentivirus), rescue group (cells infected with pLKO.1-shFoxM1 lentivirus and pLV-STMN1-GFPSpark lentivirus) and STMN1-overexpressed group (cells infected with pLKO.1-control lentivirus and pLV--GFP lentivirus). Cells were injected subcutaneously into the back of 5-year-old nude mice for 4 weeks to grow tumors. The mice were killed and the tumors were removed to measure the weight and volume. Tumor volume was measured and calculated by v = 0.5 × Length × Width2; n = 5. The data were presented as the mean ± SD. The significance was analyzed by Student’s t-test. *P < 0.05

Fig 4: FoxM1 transcriptionally activates STMN1 in cancer cells. a The expression of FoxM1 and STMN1 in six hepatocellular carcinoma cell lines, five gastric cancer cell lines, five colorectal cancer cell lines, a human hepatocyte cell line LO2 and a human gastric mucosal epithelial cell line GES-1. The gray density analysis for the data of Western blot was done by using ImageJ software, and the correlation of FoxM1 and STMN1 expression was analyzed in the above 16 cell lines, excepting an LIHC cell line SNU-739 and a CRC cell line Colo-320. b The hepatocellular carcinoma cell line SMMC-7721, gastric cancer cell lines SGC-7901 and MKN-28, colorectal cancer cell lines HCT 116 and HT-29 were used to established FoxM1-knockdown cells by lentivirus-mediated shRNA delivery system. The human hepatocellular carcinoma cell line Huh7, gastric cancer cell line AGS and colorectal cancer DLD-1 were used to establish FoxM1-overexpressed cells by lentivirus infection. The protein levels of FoxM1 and STMN1 were detected using Western blot. The qRT-PCR assay was performed to detect the mRNA expression of STMN1 in FoxM1-knockdown or -overexpressed cells. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by the Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. c Analysis of the ChIP-seq data of FoxM1 in HEK293T and K562 cells from the ENCODE database, respectively. The red box shows the binding peaks of FoxM1 on the STMN1 genomic locus. d Dual luciferase reporter assay of FoxM1 and STMN1 promoter or its mutation. The binding site of FoxM1 is in the transcription start site of -163 ~ -168. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by the Student’s t-test. *P < 0.05. e The ChIP-qPCR was used to determine the direct binding of FoxM1 on the promoter region of STMN1 in SGC-7901, HCT 116 and HT-29 cell lines. Primers located on the upstream of STMN1 transcript start site (-2893 ~ -3000) was designed for a negative control. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by the Student’s t-test. *P < 0.05

Fig 5: STMN1 is essential for survival and proliferation in cancer cells in vitro. a The hepatocellular carcinoma cell line SMMC-7721, gastric cancer cell line SGC-7901, colorectal cancer cell lines HCT 116 and HT-29 were used to establish STMN1-knockdown cells using pLKO.1 gene silence system. The hepatocellular carcinoma cell line Huh7, gastric cancer cell lines AGS were used to establish STMN1-overexpression cell lines by lentivirus infection. The protein levels of STMN1 were detected by Western blot, and the mRNA levels were detected by qRT-PCR. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. ***P < 0.001. b Cell viability of cells was detected by cell counting-8 kit (CCK-8). The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. ***P < 0.001. c Plate clone assay was performed and the number of clones was measured by ImageJ software. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. **P < 0.01, ***P < 0.001. d Soft agar assay was performed and the tumorigenic sphere was photographed after 2 weeks. The data were presented as the mean ± SD of three independent experiments. The significance was analyzed by Student’s t-test. **P < 0.01, ***P < 0.001. e Cell cycle was analyzed by flow cytometry. f Cell mitosis was analyzed by immunofluorescence of a-Tubulin. The nuclei (blue) are stained with DAPI and the a-Tubulin (red) is stained with Alexa Flour 555. The images were captured at 60× magnification, and the scale bar is 10 µm. The yellow arrows point to the normal mitosis cells. The percentage of normal or abnormal cells was calculated by numbers of normal or abnormal nucleus divided by numbers of total nucleus × 100%. The data were presented as the mean ± SD of three different fields of view at low magnification (20×). The significance was analyzed by Student’s t-test. *P < 0.05, **P < 0.01