Fig 2: Inhibition of UCHL3 weakened cancer stem cell properties, as noted in a schematic model of the effects of UCHL3 on tumorigenesis and stem-like properties. a TCID decreased AhR ubiquitination in HEK293T cells by inhibiting UCHL3 activity. AhR-Flag and Ub-His were coexpressed with vectors in HEK293T cells, and cells were treated with DMSO and TCID (10 µM) for 24 h. Cell lysates were harvested after 72 h. The AhR protein was immunoprecipitated, and polyubiquitinated AhR protein was detected by WB using anti-Ub antibody. b Western blot analysis was used to detect stemness-associated markers in UCHL3-overexpressing A549 cells treated with DMSO or TCID (10 µM). c RT-qPCR was used to detect AhR mRNA levels in UCHL3-overexpressing A549 cells treated with DMSO or TCID (10 µM) (n = 3). Data are shown as the mean ± SD; ns indicates nonsignificant (p > 0.05). d TCID accelerated AhR protein degradation. After cotreatment of A549 cells overexpressing UCHL3 with DMSO/TCID (10 µM) for 24 h and cycloheximide (CHX, 10 µg/ml) for the indicated duration, AhR protein expression was analyzed by WB. e Flow cytometry analysis showing side populations among A549 cells stably overexpressing UCHL3 treated with DMSO or TCID (10 µM) for 24 h, with the results shown as a bar graph (n = 3). Data are shown as the mean ± SD; *p < 0.05. f Representative images of flow cytometry analysis to detect CD338-positive cells among A549 cells stably overexpressing UCHL3 treated with DMSO or TCID (10 µM) for 24 hours, with the results shown as a bar graph (n = 3). Data are shown as the mean ± SD; *p < 0.05. g The MTS assay was used to assess the cell viability of A549 cells stably overexpressing UCHL3 treated with DMSO or TCID (10 µM) (n = 5). Data are shown as the mean ± SD; **p < 0.01. h Colony formation assays were performed to detect the colony formation ability of A549 cells stably overexpressing UCHL3 treated with DMSO or TCID (10 µM) (n = 3); representative images are shown on the side. Data are shown as the mean ± SD; *p < 0.05. i A549 cells stably overexpressing UCHL3 treated with DMSO or TCID (10 µM) were seeded in ultralow attachment dishes to allow tumor sphere formation, with the results shown as a bar graph (n = 5, scale bar = 100 µm). Data are shown as the mean ± SD; ***p < 0.001. Data in all bar graphs were assessed by two-tailed Student’s t-test

Fig 3: Knockdown of UCHL3 inhibited cell growth, colony formation, tumor formation, and tumor stem-like properties. a The MTS assay was used to assess cell viability in H358 cells with stable UCHL3 knockdown (n = 5). Data are shown as the mean ± SD; ****p < 0.0001. b A colony formation assay in plates was performed to detect the colony formation ability of H358 cells with stable UCHL3 knockdown (n = 3); representative images are shown in the Supplementary section, and the results show that knockdown of UCHL3 inhibited colony formation. Data are shown as the mean ± SD; **p < 0.01. c The H358 cell line with UCHL3 knockdown was seeded in ultralow attachment dishes to allow tumor sphere formation, and the results are shown as a bar graph (n = 3, scale bar = 100 µm). Data are shown as the mean ± SD; **p < 0.01. d Representative images from flow cytometry analysis to detect CD338-positive cells among UCHL3-knockdown H358 cells, with the results shown as a bar graph (n = 3). Data are shown as the mean ± SD; **p < 0.01. e Flow cytometry analysis of ALDH activity in the UCHL3-knockdown H358 cell line, with the results shown as a bar graph (n = 3). Data are shown as the mean ± SD; ****p < 0.0001. f–h A xenograft model of tumor growth was established to evaluate the ability of H358 cells with stable UCHL3 knockdown to form tumors. Tumor formation was monitored at the indicated times (f), and images (h) and tumor weights (g) were recorded (n = 6 mice per group). Data are shown as the mean ± SD; **p < 0.01, ****p < 0.0001. Data in all bar graphs were assessed by one-way ANOVA with multiple comparisons

Fig 4: UCHL3 promotes tumor stem-like properties through AhR. a Western blot analysis was used to detect AhR levels in nuclear and cytosolic fractions derived from A549 cells overexpressing UCHL3. b–d Western blot analysis was used to detect stemness-associated markers in A549 (b) and H1299 (c) cells overexpressing UCHL3 or UCHL3-knockdown H358 cells (d). e ChIP analysis was performed in A549 cells overexpressing UCHL3 to detect AhR binding to stemness-related genes as indicated (n = 3). Data are shown as the mean ± SD; two-way ANOVA with multiple comparisons; ***p < 0.001, ****p < 0.0001. f, g Western blot analysis was used to detect stemness-associated markers in (f) xenograft tumors from A549 cells overexpressing UCHL3 and (g) H358 cells with UCHL3 knockdown. h, i Western blot analysis was used to detect stemness-associated markers in UCHL3-overexpressing A549 (h) and H1299 (i) cells transiently transfected with AhR-knockdown shRNA plasmid or blank plasmid. Cell lysates were harvested at 48 h

Fig 5: UCHL3 interacts with AhR and stabilizes the AhR protein through deubiquitination. a–c Western blot analysis was used to detect the expression level of AhR in A549 (a), H1299 (b) and H358 (c) cells after overexpression or depletion of UCHL3. d Exogenous UCHL3 and AhR proteins interacted in HEK293T cells. AhR and UCHL3 were coexpressed in HEK293T cells, and the AhR protein was immunoprecipitated with anti-AhR antibody. IgG served as a negative control, and exogenous UCHL3 was detected by WB. e, f UCHL3 overexpression delayed AhR protein degradation. After the treatment of UCHL3-overexpressing A549 (e) and H1299 (f) cells with cycloheximide (CHX, 10 µg/ml) for the indicated durations, AhR protein expression was analyzed by WB. Quantification of the AhR protein band was performed using ImageJ software. g UCHL3 knockdown enhanced AhR protein degradation. After UCHL3 knockdown, H358 cells were treated with cycloheximide (CHX, 10 µg/ml) for the indicated duration, and AhR protein expression was analyzed by WB. Quantification of the AhR protein band was performed using ImageJ software. h, i The lysates of A549 (h) and H1299 (i) cells stably overexpressing UCHL3 and vector-transfected cells containing 1 mg of total protein for each panel were immunoprecipitated with 2 µg of anti-AhR antibody, following which AhR ubiquitination was examined using anti-Ub antibody. j The lysates of stable UCHL3-knockdown H358 cells and shCtrl-transfected cells containing 1 mg of total protein were immunoprecipitated with 2 µg of anti-AhR antibody, and AhR ubiquitination was examined using anti-Ub antibody