Fig 2: 5-hydroxytryptamine receptor 7 (HTR7) overexpression promotes laryngeal cancer proliferation. (A) Gene Set Enrichment Analysis of the relationship between HTR7 expression and CCND1 expression. (B) Western blot analysis of cyclin D1, cyclin E, p16, and p21 levels when HTR7 was overexpressed in FaDu cells. GAPDH was used as the loading control. (C) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of the effect of HTR7 overexpression on laryngeal cancer proliferation. (D) Colony formation assay of the effect of HTR7 overexpression on laryngeal cancer proliferation using crystal violet staining. Microscope magnification 200×. (E) BrdU incorporation assay of the effect of HTR7 overexpression on laryngeal cancer proliferation. Error bars represent the mean ± STDEV. *P<0.05.

Fig 3: Enhancing of TRIM28 was critical for SNHG8-mediated malignant behaviors. (A, B) TRIM28 expression was increased in AGS-BX1/SNHG8-OE cells and reduced by silencing of TRIM28 tested by RT- PCR and Western blot. (C) SNHG8 enhanced cell proliferation could be reduced by silencing of TRIM28 tested with MTS assay. (D) SNHG8 enhanced colony formation could be reduced by silencing of TRIM28. (E) SNHG8-enhanced cell cycle stopping at G2/M could be reduced by silencing of TRIM28 as detected by flow cytometry. (F, G) SNHG8-enhanced expression of BCL-2, CCND1, C-PARP, and PCNA could be reduced by silencing of TRIM28 tested with qRT-PCR and Western Blot. (H, I) SNHG8-enhanced cell migration could be reduced by silencing of TRIM28 tested with wound healing assay. (J) SNHG8-enhanced cell invasion could be reduced by silencing of TRIM28 tested with transwell assay. (K, L) SNHG8-enhanced expressions of invasion/metastasis-related-genes of VIM and CDH2 could be reduced by silencing of TRIM28 tested with qRT-PCR and Western blot. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 4: Let-7i-3p directly targets CCND1. (a) Putative WT and mutant‐type (MUT) let-7i-3p target sequences of CCND1 mRNA 3′-UTR. (b and c) Relative luciferase activity in HCT116 and 293T cells co-transfected with constructs carrying WT or mutant-type CCND1 mRNA 3′-UTR with let-7i-3p or NC. (d and e) The overexpression of let-7i-3p in the HCT116 cells attenuated the expression of CCND1 mRNA and protein levels, respectively, and a statistical histogram was shown at right (f). *P < 0.05 and **P < 0.01.

Fig 5: KN-93 effect on cell cycle of A549 and MSTO-211H cells. (A). The cell cycle was analyzed by FACS in A549 and MSTO-211H cells treated with 20 µM KN-93 or 20 µM KN-92 for 16 h. (B). Protein levels of cyclin D1 (CycD1) and cyclin D3 (CycD3) were assessed in A549 and MSTO-211H cells treated for 16 h with 20 µM KN-93. (C). A549 and MSTO-211H cells were co-transfected with plasmids coding for cyclin D1, CDK6-HA, CDK6-DN-HA or an empty vector as control and then treated with 12.5 µM AKTi VIII or 20 µM KN-93 for 16 h. RBL1/p107 levels were assessed by immunoblot. CDK6 was detected using an anti-HA antibody. (D). RBL1/p107 protein levels in A549 and MSTO-211H cells transduced with a lentivirus expressing CDK6 S178P or control lentivirus and transfected with plasmids coding for cyclin D3 or empty vector and then treated with 12.5 µM AKTi VIII or 20 µM KN-93 for 16 h. The expression of exogenous CDK6 S178P was monitored by detecting enhanced green fluorescent protein (EGFP), encoded by the bicistronic IRES pHAGE vector. Quantification was performed by densitometric analysis using the ImageJ program and expressed as % of CTR (B) or, for each treatment condition, as % empty vector (e. v.)-transfected respective control cells. All experiments were performed three times.