Fig 2: 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

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: The correlation of HDAC7 and FGF18 expression in NSCLC. a Kaplan–Meier survival analysis of FGF18 expression in 1925 NSCLC cases from the Kaplan–Meier plotter database. b Western blot analysis of FGF18 expression in tumor (T) and paired adjacent nontumor (N) tissues from 12 NSCLC patients. (c) Representative FGF18 IHC images of NSCLC and adjacent non-tumor tissues. Scale bar, 200 µm and 20 µm (inset), respectively. d The IHC analysis of FGF18 expression in 319 NSCLC patients. e Kaplan–Meier survival analysis of FGF18 expression in 319 NSCLC patients. f Correlation analysis of HDAC7 and FGF18 expression in NSCLC tissues. g The comprehensive Kaplan–Meier survival analysis of HDAC7 and FGF18 in 319 NSCLC patients based on the microarray tissue IHC results. h The schematic of a simplified regulatory network involving USP10 and FGF18 in HDAC7-meditated NSCLC progression. USP10 interacts with and stabilizes HDAC7 through deubiquitination, preventing its proteasomal degradation. Elevated HDAC7 expression facilitates ß-catenin redistribution to the nucleus by deacetylating ß-catenin and inhibiting its phosphorylation. As a result, the nuclear accumulation of ß-catenin binds to TCF/LEF to activate the transcription of FGF18 and thus promotes NSCLC proliferation and metastasis