Fig 2: HDAC7 regulated cell proliferation and metastasis via FGF18 in NSCLC. a The volcano plot of DEGs identified from RNA-seq data of SK-LU-1 cells overexpressing HDAC7 or the control. b Representative western blotting of FGF18 and FGFR3 in HDAC7 overexpression and knockdown cells. c Representative western blotting of whole cell lysates (WCL) at indicated times. d Representative western blotting of HDAC7, FGF18, CDK2, and Cyclin A2 in the indicated groups. e Representative images and results of colony formation assay. f Gross photograph of subcutaneous xenograft tumors, tumor growth curve and tumor weight in indicated groups. g Representative scratch wound assay images and results. h Representative western blotting of E-cadherin, Snail, and vimentin in indicated groups. i Representative fluorescence images and data analysis of pulmonary metastases 4 weeks after tail vein injection. j The general morphology of surface lung metastases. The black triangular arrows indicate the metastatic nodules. The number of metastatic nodules per lung in HE images (k) and representative HE staining images (l) from lung samples of indicated groups. Scale bar, 1 mm and 100 µm (inset), respectively. ß-actin was used as internal control. All the data are expressed as mean ± SD. *P < 0.05. LV, lentivirus

Fig 3: HDAC7 deacetylated ß-catenin at Lys49 and promoted its translocation into the nucleus to activate FGF18 expression. a HDAC7 and ß-catenin interacted in NSCLC cells. b Two-color immunofluorescence analysis showing significant colocalization of HDAC7 with ß-catenin in A549 and SK-LU-1 cells. DAPI (blue), HDAC7 (green), and ß-catenin (red). c Representative western blotting results showing HDAC7 deacetylated ß-catenin at Lys49 and phosphorylated ß-catenin at Ser45. d Representative western blotting showing upregulated HDAC7 facilitated ß-catenin nuclear translocation. e Representative western blotting of HDAC7, FGF18, ß-catenin, and TCF4 in NSCLC cells after LF3 treatment. ß-actin was used as an internal control to normalize the total protein amount. GAPDH and Lamin B1 were used as the loading control to cytosolic protein and nuclear protein, respectively. DAPI fluorescence marking the cell nucleus. LV, lentivirus. IP, immunoprecipitation. IB, immunoblot

Fig 4: USP10 interacted with and stabilized HDAC7. a USP10 and HDAC7 interacted in NSCLC cells. b The statistical analysis of relative HDAC7 mRNA levels in USP10 overexpression and knockdown cells. mRNA was normalized to GAPDH. c Representative western blotting of USP10, HDAC7 and FGF18 in indicated cells. d Protein stability analysis showing a reduced half-life of HDAC7 in USP10 knockdown cells. e Knockdown of USP10 promoted ubiquitination of HDAC7 in NSCLC cells. IP with anti-HDAC7 antibody was followed by IB with anti-ubiquitin antibody to detect polyubiquitinated HDAC7. f Representative western blotting of USP10 inhibitor indicating that Spautin-1 reduced the half-life of HDAC7. The NSCLC cells were treated with Spautin-1 at the indicated concentration and time intervals. For graphs in d and f, the relative HDAC7 levels were quantified using Image Lab software. Representative images and statistical analysis of colony formation assay (g), scratch wound assay (h), and transwell assay (i) in A549 cells after treatment with Spautin-1. Migration ability is expressed as the mean scratch area at each time point. The initial scratched area (0 h) was set as 100%. ß-actin was used as the internal control. LV, lentivirus. IP, immunoprecipitation. IB, immunoblot