Fig. 1: USP5 modulates Beclin 1 protein expression to regulate autophagy, cancer cell senescence, and lung tumor growth.

a, b Stable A549-RFP-GFP-LC3 or H292-RFP-GFP-LC3 reporter cells expressing either WT USP5 or USP5^W209A were analyzed for LC3 puncta formation under a confocal microscope. Quantification of RFP-LC3 and GFP-LC3 puncta was shown (a). A549 and H292 cells stably expressing WT USP5 or USP5^W209A were subjected to western blot (b). c–h A549 and H292 cells stably expressing shRNA specific for USP5 (#1 or #2) or control shRNA (shC) (c–e) or A549 and H292 cells stably expressing shUSP5 (shUSP5 #1), shUSP5 + WT Beclin 1 or Beclin 1^S90+93A (f–h) were subjected to western blot, SA-β-gal staining or colony formation assays. i–n For tumor growth assay, A549-Antares2 cells (2 × 10⁶) stably expressing shUSP5, shUSP5+Beclin 1 or shUSP5 + Beclin 1^S90+93A were subcutaneously transplanted into nude mice (n = 5/group). Diphenylterazine (DTZ) was injected into tumor regions in anesthetized mice and bioluminescence was subsequently imaged (i), and the harvested tumors were photographed (j), and weighted (k). The tumor volumes of cell-derived xenografts were quantified every other day (l). The tumors were subjected to immunohistochemical (IHC) staining for Ki67 and SA-β-gal (m), and the percentages of Ki67-positive cells were presented (n). o–r Human lung adenocarcinoma tissue microarrays consisting of lung tumor and adjacent normal tissues (n = 88 pairs) were subjected to IHC for USP5 and Beclin 1 (o), with quantitative analyses using average optical density (AOD) (p). The two-tailed Pearson correlation between USP5 with Beclin 1 protein levels (q) and the correlation of USP5 or Beclin 1 protein levels with Overall Survival in lung cancer patients (r) were analyzed. Experiments were performed three times independently (a–h). Data were presented as mean ± SD (a–d, f, g, p) or SEM (k, l, n). Comparisons were performed with two-way (a–d, f, g, k, l, n) ANOVA with Tukey’s test analysis for multiple comparisons and two-tailed Student’s t-test (p). Scale bar = 50 μm.