Fig 1: Deubiquitinase UCHL3 is required for JAK2-induced BRD4 stabilization.a Identification of deubiquitinases of BRD4. Purified unbiquitinated BRD4 was incubated with indicated deubiquitinases according to the manufacturer’s instructions (DUB Scan kit, Cat. No. 67-0006-001). After that, western blot analysis was performed to probe ubiquitination of BRD4 (Fig. S4a). Quantification of relative intensity of ubiquitin of BRD4 normalized to BRD4 incubated with buffer. b Heat map showing frequency of high expression of candidate DUBs in 50 paired CRC and adjacent normal mucosa tissues. c Representative western blot analysis (n = 2) of indicated proteins in PDC1 and HCT116 cells transfected with indicated siRNAs with 50 ng/ml rIL6. d Representative western blot analysis (n = 2) of indicated proteins in PDC1 cells transfected with indicated siRNAs with/without 20 µg/ml MG132 under rIL6 (50 ng/ml) treatment. e In vitro ubiquitination assays (n = 3) of WCL and immunoprecipitates from 293T cells transfected with indicated siRNA or constructs in the presence of 20 µM MG132 and 50 ng/ml rIL6. f Representative western blot analysis (n = 3) of WCL from 293T cell transfected with Flag-BRD4 or Flag-BRD4-Y97/98A together with HA-UCHL3 constructs. g Representative western blot analysis (n = 2) of indicated proteins in 293T cells transfected with indicated siRNAs or constructs. h Representative western blot analysis (n = 2) of WCL and immunoprecipitates by anti-HA antibody from 293T cells transfected with indicated constructs with 20 µM MG132 or 2.5 µM pacritinib as indicated. i Representative western blot analysis (n = 2) of WCL and immunoprecipitates by anti-HA antibody from 293T cells transfected with indicated constructs with 20 µM MG132. j Representative western blot analysis (n = 2) of WCL and immunoprecipitates by anti-BRD4 antibody from PDC1 cells treated with 50 ng/ml rIL6 and/or 2.5 µM pacritinib as indicated.
Fig 2: UCHL3 maintained the stability of AhR protein through deubiquitination, thereby promoting PD-L1 expression. A, Positive expression of AhR protein in tumor tissues and adjacent normal tissues from NSCLC patients (n = 45) detected by Immunohistochemistry. B, Silencing efficiency of sh-UCHL3 in A549 cells and overexpression efficiency of LV-UCHL3 in PC9 cells detected by qRT-PCR. C, Protein levels of UCHL3 and AhR in A549 cells treated with sh-UCHL3 or in PC9 treated with LV-UCHL3 detected by Western blot analysis. D, Western blot analysis of AhR protein degradation in A549 and PC9 cells following CHX (10 µg/mL) treatment (the left penal) and corresponding statistical analysis of AhR protein stability (the right penal). E, Co-IP detection of exogenous UCHL3 and AhR proteins in HEK293T cells. F-G, AhR protein degradation rate in A549 cells in response to sh-UCHL3 (F) or in PC9 cells in response to LV-UCHL3 (G) detected by CHX pulse-chase analysis. H, Detection of AhR protein ubiquitination in A549 cells overexpressing UCHL3 or in PC9 cells silencing UCHL3. I, Expression of PD-L1 in tumor tissues and adjacent normal tissues from NSCLC patients (n = 45) detected by qRT-PCR. J, Expression of PD-L1 in A549 cells treated with sh-UCHL3 or in PC9 cells treated with LV-UCHL3 detected by qRT-PCR. K, Silencing efficiency of sh-AhR in A549 cells and overexpression efficiency of LV-AhR in PC9 cells detected by qRT-PCR (the left two panels) and Western blot analysis (the right two panels). L, Expression of PD-L1 in A549 cells treated with sh-AhR or in PC9 cells treated with LV-AhR detected by qRT-PCR. M, Protein levels of UCHL3, AhR and PD-L1 in A549 cells treated with sh-UCHL3 or combined with LV-AhR or in PC9 cells treated with LV-UCHL3 or combined with sh-AhR detected by Western blot analysis. * p < 0.05 versus the adjacent normal tissues or A549 cells treated with sh-NC; # p < 0.05 versus the PC9 cells treated with Vector; & p < 0.05 versus the A549 cells treated with sh-UCHL3 + Vector; @ p < 0.05 versus the PC9 cells treated with LV-UCHL3 + sh-NC. Each cellular experiment was repeated 3 times
Fig 3: Graphical summary of the regulatory mechanism of LINC00665 in NSCLC radiosensitivity. LINC00665 acted as a sponge of miR-582-5p to inhibit miR-582-5p expression, up-regulated the expression of UCHL3, and enhanced the stability of AhR protein, thereby promoting the immune escape and reducing the radiosensitivity in NSCLC cells
Fig 4: LINC00665 augmented the immune escape of NSCLC cells through the miR-582-5p/UCHL3 regulatory axis by mediating the stability of AhR protein. A, Representative images of xenografts in tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR. B, Tumor weight of the xenografted tumor in tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR. C, qRT-PCR determination of miRNA levels of LINC00665, miR-582-5p, UCHL3, AhR, and PD-L1 in tumor tissues from tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR. D, Western blot analysis of protein levels of UCHL3, AhR, and PD-L1 in tumor tissues from tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR. E, Detection of ki67-positive cells was to assess tumor cell proliferation in tumor tissues from tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR. F, Cell apoptosis in tumor tissues from tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR determined by TUNEL staining. G, Representative immunofluorescence images of CD8, PD-1, D240 and PD-L1 in tumor tissue sections (the left panels) and corresponding statistical analysis (the right panel). H, I, ELISA detection of INF-? (H) and TNF-a (I) in tumor tissues from tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR. J, qRT-PCR measurement of CXCL10 expression in tumor tissues from tumor-bearing mice under irradiation treatment and injected with PC9 cells overexpressing LINC00665 alone or in combination with silencing AhR. n = 10. * p < 0.05 versus the mice treated with PC9/Vector; # p < 0.05 versus the mice treated with PC9/Vector + Gy; & p < 0.05 versus the mice treated with PC9/LV-LINC00665 + sh-NC + Gy
Fig 5: UCHL3 reduced the radiosensitivity of NSCLC cells by stabilizing AhR protein. A, Cell viability in A549 cells silencing UCHL3 and in PC9 cells overexpressing UCHL3 measured by CCK-8 assay. B, Colony formation potential of A549 cells treated with sh-UCHL3 or in PC9 cells treated with LV-UCHL3 measured by colony formation assay. C, Cell invasion in A549 cells treated with sh-UCHL3 or in PC9 cells treated with LV-UCHL3 measured by Transwell assay. D, Detection of ?H2ax fluorescence in A549 cells treated with sh-UCHL3 or in PC9 cells treated with LV-UCHL3. E, Flow cytometry analysis of cell apoptosis in A549 cells treated with sh-UCHL3 or in PC9 cells treated with LV-UCHL3. F, Flow cytometry analysis of cell cycle distribution in A549 cells treated with sh-UCHL3 or in PC9 cells treated with LV-UCHL3. G, Cell viability in A549 cells treated with sh-UCHL3 or combined with LV-AhR or in PC9 cells treated with LV-UCHL3 or combined with sh-AhR measured by CCK-8 assay. H, Colony formation potential of A549 cells treated with sh-UCHL3 or combined with LV-AhR or in PC9 cells treated with LV-UCHL3 or combined with sh-AhR measured by colony formation assay. I, Cell invasion in A549 cells treated with sh-UCHL3 or combined with LV-AhR or in PC9 cells treated with LV-UCHL3 or combined with sh-AhR measured by Transwell assay. J, Detection of ?H2ax fluorescence in irradiation-treated A549 cells treated with sh-UCHL3 or combined with LV-AhR or in PC9 cells treated with LV-UCHL3 or combined with sh-AhR. K, Flow cytometry analysis of cell apoptosis in A549 cells treated with sh-UCHL3 or combined with LV-AhR or in PC9 cells treated with LV-UCHL3 or combined with sh-AhR. L, Flow cytometry analysis of cell cycle distribution in A549 cells treated with sh-UCHL3 or combined with LV-AhR or in PC9 cells treated with LV-UCHL3 or combined with sh-AhR. *, # p < 0.05. Each cellular experiment was repeated 3 times
Supplier Page from Abcam for Anti-UCHL3 antibody