Fig 2: TMEM100 is downregulated in CRC tissues and reduced TMEM100 expression indicated detrimental prognosis. (A) Relative mRNA expression level of TMEM100 in 23 CRC tissues and paired adjacent tissues was evaluated by RT-qPCR. Two-sided t-test. (B) Western blotting analysis of TMEM100 in 6 paired adjacent tissues and CRC tissues. β-actin was used as a reference control. Two-sided paired t-test. (C) Western blotting analysis of TMEM100 level in six human CRC cell lines and two human normal intestinal epithelial cell lines (***p < 0.001 vs NCM460). β-actin was used as a reference control. Two-sided t-test. (D) Representative images of IHC staining of TMEM100 in 186 CRC tissues and 164 adjacent tissues in the tissue microarray. Scale bar = 50 μm. Two-sided t-test. (E, F, G) Overall survival was defined as the interval between the date of surgery and the date of death or last follow-up. The CRC patients (n=101) with lower TMEM100 level indicated detrimental prognosis (E). The CRC patients (n=101) with positive regional lymph node metastasis indicated detrimental prognosis (F). The CRC patients (n=101) with both lower TMEM100 level and positive regional lymph node metastasis indicated worse prognosis (G). Data are presented as the mean ± SD. **p<0.01, *** p<0.001.

Fig 3: TMEM100 restrains the migration and invasion capacities of CRC cells. (A, B) Transwell assays were performed to evaluate the capacities of migration and invasion in indicated cells. Scale bar = 100 μm. Two-sided t-test. (C) Western blotting analysis of E-cadherin and Vimentin expression in indicated cells. β-actin was used as a reference control. Two-sided t-test. (D) The representative pictures of the metastatic neoplasm in the lung of the mice metastasis model (n=5/group). The section which presents the largest number of CRC cells colonized foci was selected, and the yellow arrow represents the foci of which diameter was a minimum of 1.5 μm. Bar in the left picture represents 5 μm, and bar in the right picture represents 1.43 μm. Two-sided t-test. Data are presented as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

Fig 4: TMEM100 inhibits proliferation of CRC cells in vitro and in vivo. (A) CCK-8 assays were used to detect the impact of TMEM100 on the proliferation rate of CRC cells. Two-sided t-test. Red asterisks represent si-NC vs. si-TMEM100-1. Blue asterisks represent si-NC vs. si-TMEM100-2. (B) Colony formation assay was used to detect the impact of TMEM100 on the colony formation capacity of CRC cells. Two-sided t-test. (C) General observation of subcutaneous tumors in nude mice (n = 5/group) were displayed. (D) The weight of subcutaneous tumors in nude mice (n = 5/group) was analyzed. Two-sided t-test. (E) The growth curve of the subcutaneous tumors (n=5/group). Two-sided t-test. (F) The representative pictures of the subcutaneous tumors stained by HE and the Ki67 expression stained by IHC (n=5/group). Scale bar = 100 μm. Two-sided t-test. All data are presented as the mean ± SD. *p<0.05, **p<0.01, *** p<0.001.

Fig 5: TMEM100 overexpression leads to a reduction in HIF-1α content and microvascular density in CRC. (A) Representative images of IHC staining of TMEM100 and HIF-1α in CRC tissues. Scale bar = 100 μm. (B) The correlation between TMEM100 and HIF-1α expression in CRC specimens was analyzed, and the linear correlation coefficient and statistical significance were indicated. (C) HCT116 cells were infected with LV-vector or LV-TMEM100 lentiviruses. The relative mRNA expression of HIF-1α was detected by RT-qPCR in indicated cell under normoxic and hypoxic conditions. ANOVA. (D) TMEM100 and HIF-1α expression in indicated cell was detected by western blotting. β-Actin was used as a reference control. Two-sided t-test. (E) The extracellular concentration of VEGF released by indicated cells was detected by ELISA. ANOVA. (F) The representative pictures of the HIF-1α and CD34 expression of subcutaneous tumors (n=5/group) stained by IHC. Two-sided t-test. (G) Control or TMEM100 overexpression cells were incubated with cycloheximide (CHX, 50 μg/mL) for the indicated time. The cell lysates were then analyzed by western blotting. The dot lines indicated the half-life of the HIF-1α. β-actin was used as a reference control. Two-sided t-test. Data are presented as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001. NS, no significance.