Fig 2: Effects of circ-MTCL1 on proliferation, invasion and migration through C1QBP in LSCC cells. a, The expression level of circMTCL1 was identified after overexpressing or knocking down circMTCL1 by qRT-PCR assays. Values are the mean ± s.d. of n = 3 independent experiments. b, Western blot assays were performed to evaluate the expression levels of ß-catenin and p-ß-catenin upon circMTCL1 silencing or overexpressing in TU212 and LCC cells. c, The cytoplasmic and nuclear accumulation of ß-catenin and p-ß-catenin were determined after ectopic or knockdown circMTCL1 by Wertern blot assays. d, The proliferation ability was measured upon circMTCL1 overexpressing or silencing in TU212 and LCC cells by EdU assays. Scale bars = 50 µm. e, Wound healing assays were performed to identify the cell motility upon circMTCL1 silencing or overexpressing in TU212 and LCC cells. f, The migrated cell numbers were determined after ectopic or knockdown circMTCL1 in TU212 and LCC cells. g,Western blot assays detected the expression of ß-catenin and p-ß-catenin in TU212 and LCC cells co-transfected circMTCL1 and C1QBP. h, CircMTCL1 rescued the proliferation ability after co-transfected circMTCL1 and C1QBP. i, Overexpressing circMTCL1 rescued the vertically migrated and invasive ability after silencing C1QBP. Scale bars = 500 µm. J, Overexpressing circMTCL1 rescued the laterally migrated ability after silencing C1QBP. Scale bars = 500 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig 3: The mechanisms underlying circMTCL1 promoted LSCC progression: circMTCL1 binds to C1QBP to inhibit the degradation of C1QBP through viaubiquitin-proteasome pathway subsequently activating the wnt/ß-catenin pathway

Fig 4: C1QBP modulates XDH mRNA at translational level. The XDH pre-mRNA was examined by qRT-PCR in control group and C1QBP knockdown or overexpression group of ACHN (A) and 786-O (B) cells. TNF-α, IL6, IL-1β, and IFN-γ mRNA were examined by qRT-PCR in control group and C1QBP knockdown or overexpression group of ACHN (C) and 786-O (D) cells. (E) Control and C1QBP overexpressed ACHN and 786-O cells were treated with 5 μg/ml actinomycin D at indicated time point 0 min, 15 min, 30 min, 45 min, and 60 min. The RNA was extracted and XDH mRNA was detected by qRT-PCR. Statistically significant differences were indicated: *, P < 0.05, **, P < 0.01 and ***, P < 0.001. NS: no significant difference.

Fig 5: XDH is critical for C1QBP-regulated ROS production and apoptosis of RCC. XDH knockdown by using three independent XDH siRNAs (si-XDH-1, si-XDH-2, si-XDH-3) in ACHN and 786-O cells were evidenced by qRT-PCR (A) and western blot (B). ACHN and786-O cells were transfected with pCDH + si-NC, pCDH-C1QBP + si-NC, pCDH-C1QBP + si-XDH, and then ROS level (C and D) and apoptosis (E and F) were examined by flow cytometry, and (G) the expression of C1QBP, XDH, cleaved-caspase-3, bcl2, and bax was examined by western blot. β-actin was used as an internal control. Data were presented as mean ± SD by t-test. Statistically significant differences were indicated: *, P < 0.05, **, P < 0.01 and ***, P < 0.001. NS: no significant difference.