Fig 2: NUPR1 binds to AhR and promotes degradation of AhR in the autophagy-lysosome pathway. a Western blot was used to examine the protein levels of AhR, ARNT, and HSP90 in NUPR1-overexpressing MHCC-97H/MHCC-97L cells and NUPR1-knockdown QGY-7701/Hep3B cells with or without IR (8 Gy). b Immunofluorescence staining was performed to determine the location of AhR (green) in the indicated cell lines. The nuclei were counterstained with DAPI (blue). Scale bar: 10 µm. c Western blot was used to detect AhR levels in indicated cell lysates collected at different timing (0, 3, 6, 9 h) after 20 µg/mL CHX treatment. d Western blot of AhR protein was performed in cell lysates from different NUPR1 expression status cell lines with or without 20 µM CQ treatment. e Representative immunofluorescence images show the distribution of AhR (red), LAMP1 (green), and DAPI (blue) in LV-NC and LV-NUPR1 MHCC-97L cells. Scale bar: 10 µm. f The levels of LC3-II and p62 in cells with a different NUPR1 expression were determined by western blot. g The anti-Flag and anti-IgG products after incubating with lysates from MHCC-97H/MHCC-97L ectopically expressed Flag-tagged NUPR1 were used to detect Flag-NUPR1 and AhR protein by western blot. h Western blot analysis of NUPR1 and AhR in anti-AhR and anti-IgG products was performed. Cell lysates were immunoprecipitated with antibodies against AhR or IgG. i Representative immunofluorescence images of QGY-7701/MHCC-97H cells show the staining of NUPR1 (red), AhR (green), and nuclei counterstained with DAPI (blue) (left). Scale bar: 10 µm. Pearson correlation of the signal intensity of indicated molecules in different subcellular locations was quantified by ImageJ software (N/nucleus, C/cytoplasm, right)

Fig 3: CD146 expression was dependent on aryl hydrocarbon receptor (AhR)/ARNT signaling. (A) Immunofluorescence analysis revealed that AhR was translocated into the nucleus in MLE-12 cells treated with PM2.5, control: MLE-12 cells treated with PBS for 12 h; PM2.5: MLE-12 cells treated with 10 µg/ml PM2.5 for 12 h. (B) Western blot analysis of AhR, ARNT, and ARNT2 in MLE-12 cells treated with 10 µg/ml PM2.5. (C) Western blot analysis of CD146 in MLE-12 cells pretreated with 1 µm AHR inhibitor (PDM2, MedChemExpress, HY-112629) for 1 h and challenged with 10 µg/ml PM2.5 for 12 h. *P < 0.05; **P < 0.01; ns, not significant.

Fig 4: Western blot analysis of AhR, ARNT and CYP1A1 in PM2.5-exposed SZ95 sebocytes. Cell extracts were subjected to 10% SDS-PAGE and western blot analysis with a primary antibody against AhR, ARNT and CYP1A1, respectively. GAPDH protein was used as an internal control. (A) Western blot analysis results of AhR, ARNT and CYP1A1. (B) Quantification of AhR, ARNT and CYP1A1 protein expression. *P<0.05 and **P<0.01 vs. control. AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator protein; CYP1A1, cytoplasmic cytochrome P4501 A1; PM, particulate matter; NW-PM2.5, non-water-soluble extracts; W-PM2.5, water-soluble extracts; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.