Fig 2: HCF-1 regulates CDC42 expression during different stages of the cell cycle.Flow cytometry analysis of synchronized cell pools. a A schematic graph of the synchronization and flow cytometry analysis procedures. The first 15 h thymidine block was performed following 9 h of thymidine release, and cells were harvested at different time points (0, 2, 4, 8 h) after the second 15 h double-thymidine block. One group of synchronized cells was treated with nocodazole 6 h after the second release and harvested after another 6 h for analysis. b Synchronized cell pools from different time points were analysed to evaluate their cell cycle distributions (n = 3). c Cell-cycle-associated changes in the mRNA expression of HCF-1 and CDC42 (n = 3). The expression levels of HCF-1 and CDC42 in synchronized cell pools were detected by semi-quantitative RT-PCR. The band intensity values relative to the control were measured with ImageJ software, and the number represents the ratio. d Cell-cycle-associated changes in the protein levels of HCF-1, CDC42, RhoA and Rac1. The expression levels of HCF-1 and CDC42 in the synchronized cell pool were detected by immunoblotting (n = 3). β-actin was used as a loading control, and E2F1 was used as a marker of G1/S phase. The band intensity values relative to the control were measured with ImageJ software, and the results are shown below the blot. e qRT-PCR analysis of the mRNA expression of HCF-1 and CDC42 at different time points. The comparisons performed were to the respective 0 h controls. Results are expressed as mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001. Student’s t-test). f ChIP analysis of HCF-1 protein on the −881 to −575 bp region of the CDC42 promoter throughout the cell cycle (n = 3). Absolute quantitative analysis of the content of templates immunoprecipitated with an HCF-1 antibody in different synchronized cell pools (0 h for G1, 4 h for S, and 6 h for the nocodazole-treated cell pool for M phase). Genomic DNA from HeLa cells was serially diluted to generate standard products to establish a standard curve of the relative threshold cycle (Ct). Then, the amount of DNA amount in the ChIP assay was quantified by qRT-PCR according to the standard curve and calculated based on input DNA. Results are expressed as mean ± SD of % input.

Fig 3: Schematic diagram of pro-metastatic function of miR-4646-5p/ABHD16A/lyso-PS axis in Drosha-low expressed gastric tumor.In Drosha-low expressed gastric cancer cells, mirtronic miR-4646-5p was abnormally up-regulated. The Elevated mirtronic miR-4646-5p is a specific splicing product of intron-3 of the host gene Abhd16a with the aid of enhanced SRSF2. The enhanced miR-4646-5p can mitigate ubiquitination of HIF1A by inhibiting the target gene PHD3, thus lead to an increased HIF1A. As a transcription factor, HIF1A feedback promotes the expressions of the host gene Abhd16a and miR-4646-5p itself. The Abhd16a encodes a lipase enzyme ABHD16A, which causes accumulation of lipid metabolite lyso-PS, and then activates RhoA through GPR34/Gi. As synergistic regulation, miRNA-4646-5p can also up-regulate RhoA expression through transcription factor HIF1A. The activation and up-regulation of RhoA synergistically promote gastric cancer metastasis through LIMK/cofilin signaling.

Fig 4: DnaJA4-KO up-regulated the expression of F-actin and related pathway proteins. (A) Western blotting analysis was used to evaluate F-actin, RhoA, ROCK1 E-cadherin, β-catenin expression. (B) Unheated (37°C) vs. hyperthermia (44.0°C) treated WT cells or DanJa4-KO cells as determined at 6, 12, and 24 h following treatment, respectively. ∗P < 0.05, †P < 0.01, ‡P < 0.001. WT: Wild-type cells; DnaJA4-KO: DnaJA4-knockout cells; GADPH: Glyceraldehyde-3-phosphate dehydrogenase; ROCK1: rho-associated serine/threonine kinase 1.

Fig 5: Small GTP‐binding protein pathways and Notch signal transduction are activated by matrix with high stiffness. A, Real‐time PCR analyses of Ras, MAP kinase‐ERK kinase (Mek) and Ras homologue family member A (RhoA) expression; B, Western blot analyses showing protein expression of Ras, Mek and RhoA; C, The protein expressions of Ras, Mek and RhoA were quantitated, and data are shown as a histogram. Each experimental value is expressed as the mean ± standard deviation; D, Real‐time PCR analyses of Notch1, hairy/enhancer‐of‐split related with YRPW motif 1(Hey1) and vascular endothelial growth factor receptor 3 (VEGFR3) expression; E, Western blotting of Notch1, Hey1 and VEGFR3; F, Protein expressions of Notch1, Hey1 and VEGFR3 were quantitated and data are shown as a histogram. *P < 0.05, **P < 0.01, ***P < 0.001. Data shown are representative of three independent experiments