Fig 2: The effect of circMTCL1 on LSCC biological functions by regulating C1QBP ubiquitination. a, Silver staining images of PAGE gels, in which circMTCL1/proteins complexs from pull down experiments of TU212 cells were separated; the arrows represent the specific protein bands in pull down complexs by circMTCL1 sense sequence when compared with antisense sequence. b, Western blot assays for SDS-PAGE electrophoresis of RNA pull-down assay. c, The peak map of C1QBP acquired from the RNA pulldown mass spectrometry assay. d, KEGG analysis of the protein C1QBP, and the related signaling pathways. e, Western blot assays showing the effect of circMTCL1 on the proteins pulled down by target RNA probe (circMTCL1). f, The C1QBP expression was conducted after overexpressing or knocking down circMTCL1 by Western blot. g, CircMTCL1 expression was determined after overexpressing, knocking down C1QBP by qRT-PCR assays . Values are the mean ± s.d. of n = 3 independent experiments. h, Reducing the translation activity of C1QBP by silencing circMTCL1 treated by the CHX in TU212 cells (left). The quantification of C1QBP degradation rate by gray scale analysis (right). i, Western blot showing C1QBP protein in TU212 cells after knocking down circMTCL1 and then treated with MG132 for 24 h. j, Ubiquitinated C1QBP measured by immunoprecipitation with anti-C1QBP antibody or IgG control and immunoblotting with anti-ubiquitin antibody in TU212 cells after knocking down circMTCL1. k, IHC method was used to determine the cell localization and the expression levels of C1QBP in LSCC tissues and matched adjacent-tumor controls (n = 140). Scale bars = 100 µm. l, Violin charts illustrating the expression scores and levels of C1QBP between LSCC tissues and corresponding adjacent normal tissues (n = 140). Nonparametric tests and Median and 95%CI were shown. m, Kaplan-Meier curves displaying the impact of C1QBP on overall survival in the included LSCC cohort. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig 3: Identification of the direct interaction between circMTCL1 and C1QBP protein in LSCC cells. a, Schematic diagram of binding manner for circMTCL1 and C1QBP. b, Co-localization of circMTCL1 with C1QBP protein detected by ISH and IF assays in TU212 and LCC cells. Scale bars = 5 µm. c, Bioinformatics online software predicting the specific binding sequence and sites of circMTCL1 secondary structure and C1QBP protein (http://www.tartaglialab.com/). d, The probe information of circMTCL1 in pull-down assay. e, RNA pull-down followed by Western blot was conducted to examine the direct interaction of the circMTCL1-WT, circMTCL1-Mut and antisense RNA probes with C1QBP protein after circMTCL1 knockdown in TU212 cells. f, RIP assay was performed to verify the combination of circMTCL1 and C1QBP in TU212 cell. g, ISH and IHC methods were used to determine the cell localization and expression levels of circMTCL1 and C1QBP in our included clinical LSCC tissues and matched adjacent-tumor controls (n = 140). Scale bars = 100 µm. h, Violin charts displaying the expression scores of circMTCL1 and C1QBP between LSCC tissues and matched paired adjacent normal tissues (n = 140). Nonparametric tests and median and 95%CIs were shown. i, Linear correlation pattern representing a positive association of circMTCL1 expression with C1QBP expression

Fig 4: CircMTCL1 promotes growth and metastasis of LSCC xenograft in vivo. a, Schematic diagram of xenografts in BALB/c nude mice by inoculating TU212 cells that were stably co-transfected with sh-NC, sh-circMTCL1, respectively, at their back. Then half of the xenografts were sacrificed at the 28th day after injection and the other half were sacrificed at the 56th day after injection. b, Representative images of subcutaneously injecting nude mice and xenograft tumors. c, In vivo image of nude mice treated with subcutaneous injection of TU212 cells stably transfected with sh-circMTCL1 or sh-NC. d, Mean tumor volumes on different days for each group xenografts in nude mice. Data are showed as mean ± s.d, n = 5 for each group. e, Mean tumor weight of each group xenografts in nude mice. Data are showed as mean ± s.d, n = 5 for each group. f, In vivo image of xenografts formed by vein injection of TU212 cells stably transfected with sh-circMTCL1 or sh-NC. g and h, HE staining of tumors and lung metastases. Scale bars = 100 µm (upper) and 500 µm (lower). Data are showed as mean ± s.d, n = 5 for each group. i, IHC assay showing the protein expression of Ki-67 in tumors and C1QBP with ß-catenin in lung sections of each group. Scale bars = 50 µm. j, ISH and IHC methods were used to identify the cell localization and expression levels of circMTCL1 and C1QBP in each group, in subcutaneous tumor and lung metastatic tumor. n = 5 for each group. Scale bars = 100 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig 5: 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