Fig 2: m6A modification of TMBIM6 mRNA enhances TMBIM6 stability through IGF2BP3-dependent. a Western blot was performed to investigate the expression of TMBIM6 and IGF2BP3 in AMC-HN-8 cells after transfection with shTMBIM6–2 and/or IGF2BP3. b Western blot was conducted to examine the expression of TMBIM6 and IGF2BP3 in TU-212 cells after transfection with shIGF2BP3–1 and/or TMBIM6. c Transwell assay was performed to measure the cell invasion ability of AMC-HN-8 cells after transfection with shTMBIM6–2 and/or IGF2BP3. Hoechst staining assay was performed to measure cell apoptosis of AMC-HN-8 cells after transfection with shTMBIM6–2 and/or IGF2BP3. d Transwell assay was performed to measure the cell invasion ability of TU-212 cells after transfection with shIGF2BP3–1 and/or TMBIM6. Hoechst staining assay was performed to measure cell apoptosis of TU-212 cells after transfection with shIGF2BP3–1 and/or TMBIM6. e-h RNA stability assay showed that different transfection groups have a different effect on the half-life of TMBIM6 mRNA

Fig 3: M6A regulator IGF2BP3 was highly expressed in SC tissues. (A) Global m6A methylation level in total RNA was measured in 20 pairs of SC tumor tissues and adjacent normal tissues. (B) Expression heatmap of 24 m6A-related genes in 303 cases of SC tumor tissues and 32 cases of normal tissues. (C) Violin plots of 24 m6A-related genes in 27 pairs of SC tumor tissues and adjacent normal tissues. Violin plots were drawn using the bioinformatics website (http://www.bioinformatics.com.cn/plot_basic_vertical_horizontal_violin_plot_068). (D) Expression analysis of IGF2BP3 in SC tumor tissues (n=408) and normal tissues (n=211). The figure was drawn by the GEPIA website (http://gepia.cancer-pku.cn/). (E) Expression level of IGF2BP3 in 6 pairs of SC tumor tissues and adjacent normal tissues was measured by western blot assay. * p < 0.05, ** p < 0.01.

Fig 4: Effect of the five clinical drugs on globular C1q receptor (gC1qR) and IGF2BP3. (A) The gC1qR and IGF2BP3 protein expression levels of gC1qR and IGF2BP3 after adding five clinical drugs to H929 and U266 for 24-h incubation tested by Western blot (WB). (B) The gC1qR protein expression levels after adding five clinical drugs separately to H929 for 24-h incubation tested by flow cytometry (FC) [fluorescein isothiocyanate (FITC) fluorescence)]. The value of mean fluorescence intensity (MFI) of FITC for each representative image is above each graph. (C) The gC1qR protein expression levels after adding five clinical drugs separately to U266 for 24-h incubation tested by FC (FITC fluorescence). The value of MFI of FITC for each representative image is above each graph. (D) Average MFI of FITC fluorescence (gC1qR) after adding five clinical drugs separately to H929 and U266 for 24-h incubation tested by FC. Error bars represent mean ± SD.

Fig 5: Globular C1q receptor (gC1qR) interacted with IGF2BP3, which also suppressed the multiple myeloma (MM)-inhibiting role of C1q. (A) Venn diagram: detection of proteins interacting with gC1qR using coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). (B) Western blot (WB) verified that gC1qR and IGF2BP3 were indeed combined with each other. Keeping the amount of INPUT consistent, the amount of gC1qR-bound IGF2BP3 in the C1q-treated group was slightly increased compared with that in the bovine serum albumin (BSA)-treated group. (C) The levels of IGF2BP3 mRNA of BM CD138+ cells in the 1q21 (+) patient group were significantly higher than those in the 1q21 (–) group (p = 0.001). Error bars represent mean ± SD. (D) Knockdown of IGF2BP3 (IGF2BP3 KD) in H929 and U266 cells was verified by WB. (E) The EDU assay tested by flow cytometry (FC) [fluorescein isothiocyanate (FITC)] between the NC group and the IGF2BP3 KD in H929 and U266 cells. The value of mean fluorescence intensity (MFI) of FITC for each representative image is above each graph. (F) Based on the data of EDU assay, the average MFI of FITC (EDU) and mean inhibition ratios of proliferation (IRPs) on each group were measured. Error bars represent mean ± SD. [1] The average MFI of FITC (EDU) in the NC group on each cell line. The mean IRPs of C1q for H929 and U266 was 33.6 and 67.9%, separately [2] The average MFI of FITC (EDU) in the IGF2BP3 KD group on each cell line. The mean IRPs of C1q for H929 IGF2BP3 KD and U266 IGF2BP3 KD were 70.7 and 73.8%, respectively. (G) Based on the data of EDU assay, with the paired-sample t test for IRPs of C1q on H929 and U266 cells at each respective time (p = 0.027). (H) The cell survival among the NC group and the IGF2BP3 KD in H929 and U266 cells by Cell Counting Kit-8 (CCK-8) (p < 0.001). Error bars represent mean ± SD.