Fig 2: TSPAN8 promotes SHH-induced SMO phosphorylation. a, b Flag-PTCH1 (a) or Flag-SMO (b) was immunoprecipitated from MCF7 cells with or without TSPAN8 overexpression in the presence or absence of ATXN3 shRNA. Cells were treated with 100 ng/ml SHH for 6 h. Immunoblotting analyses were performed with the indicated antibodies. PLVX, lentiviral stable transfection plasmid pLVX-HA-IRES-Puro. c Endogenous SMO was immunoprecipitated from MCF7 cells with or without TSPAN8 overexpression in the presence or absence of ATXN3 shRNA. Cells were treated with 100 ng/ml SHH for 6 h. Immunoblotting analyses were performed with the indicated antibodies. P-Ser, phosphorylated serine. d MCF7 cells with or without overexpressing ATXN3 or TSPAN8 or MCF7 cells with overexpressing TSPAN8 and ATXN3 shRNA were left untreated or treated with 100 ng/ml SHH. Immunofluorescent studies were performed with the indicated antibodies. e MCF7 cells with or without overexpressing ATXN3 or TSPAN8 or MCF7 cells with overexpressing TSPAN8 and ATXN3 shRNA were transfected with a GLI1 promoter–luciferase reporter plasmid. These cells were left untreated (left) or treated (right) with 100 ng/ml SHH. Luciferase activity was measured and normalized to Renilla luciferase activity (n = 3 per group). Two-tailed unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001. f, g MCF7 cells with or without expressing GFP-TSPAN8 in the presence or absence of ATXN3 siRNA expression were treated with 20 µM Paroxetine hydrochloride (f) or 2 µM GSK180736A (g) together with 100 ng/ml SHH for 6 h. Cellular extracts were immunoprecipitated with anti-SMO antibody, then immunoblotting analyses tested the serine-phosphorylation expression level. h Histograms show the mean numbers and diameters of spheres formed by MCF7 cells expressing the indicated proteins and ATXN3 shRNA. i MCF7 cells expressing the indicated proteins and ATXN3 shRNA were treated with ADR (left) or PTX (right) with the different concentrations for 24 h. The cell viabilities were determined. Cell numbers with no drug was used as control (n = 3 per group). Two-tailed unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 3: Combination of miR-619-5p and si-ATXN3 treatment showed enhanced ATXN3 inhibition in vitro. (a) HN6/CDDPR and CAL27/CDDPR were transfection with miR-619-5p and si-ATXN3 alone and combination of them, and the cell viability was detected by CCK8 assay. (b and c) Representative images showed that the effect of different combination treatments on HN6/CDDPR and CAL27/CDDPR migration and invasion. (d) Real-time RT-PCR and (e) western blotting analyses were performed to measure the expression of ATXN3, PI3K and AKT after treatment with si-ATXN3 or a combination of both.

Fig 4: A group of ER-associated DUBs are differentially ubiquitinated in response to articular cartilage injury.A, heat map of differentially ubiquitinated peptides of DUB proteins in response to injury (30 min) compared to uninjured control ranked by log2 fold of change(LogFC30 min) in three biological replicates. Statistics by unpaired t test. B, a summary of the validation approach used to confirm ubiquitomics analysis. C–E, porcine cartilage was explanted to induce injury and either snap frozen(0 min) or cultured at 37 °C for 30 min. Cartilage was then lysed as described in Experimental Procedures. Ubiquitinated proteins were enriched using anti-ubiquitin–conjugated agarose beads (Ub-IP) and agarose beads were used as a control (IP CTRL). After immunoprecipitation, immunocomplexes were analyzed by Western blotting using target protein–specific antibodies for DUB proteins in (C) YOD1 and (D) ATXN3, USP5, and BRCC3. Arrows indicate the enriched specific proteins. Shown are the blots representative of three biological replicates. E, protein levels of candidate proteins were analyzed in input tissue lysates samples. ß-actin was used as an internal loading control. Numbers on blots reflect protein molecular weight. DUB, deubiquitinase; ER, endoplasmic reticulum.

Fig 5: TSPAN8 stabilizes the expression of PTCH1 by recruiting ATXN3. a MCF7 cell lysates were immunoprecipitated with control rabbit IgG or anti-TSPAN8 and immunoblotted with anti-SHH or anti-PTCH1 antibodies. Ten percent of the cell extracts was loaded as an input. b GST pulldown assay was performed by mixing purified GST-TSPAN8 or GST with the lysates of HEK293T cell expressing His-SHH or His-PTCH1-CTD (C-terminal domain of PTCH1), then immunoblotting analyses were performed (top panel). Coomassie blue staining of GST-TSPAN8 and GST was performed (bottom panel). c Immunofluorescence analysis of MCF7 cells with or without TSPAN8 overexpression was performed. DAPI was used to stain the nuclei. Scale bar = 30 µm. d MCF7 cells with or without TSPAN8 overexpression were treated with CHX (100 µg/ml) for different periods of time. Quantitation was done by densitometry with Image J Software. SHH and PTCH1 band intensities were normalized according to the levels of GAPDH, SHH, and PTCH1 in the CHX-untreated cells (n = 3 per group, right). e MCF7 cells transfected with control siRNA or TSPAN8 siRNA were treated with 100 µg/ml CHX for the indicated periods of time then immunoblotting analyses were performed with the indicated antibodies. f MCF7 cells transfected control siRNA or TSPAN8 siRNA followed by treatment with or without MG132 (20 µM, 8 h). Immunoblotting analyses were performed with the indicated antibodies. g A vector expressing Flag-ATXN3 was co-transfected with a vector expressing GFP or GFP-TSPAN8 in HEK293T cells. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. h HEK293T cells were transfected with control siRNA or TSPAN8 siRNA together with a Flag-ATXN3 plasmid. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. i, j MCF7 cells expressing Myc-ATXN3 (i) or ATXN3 shRNA (j) were treated with MG132 (20 µM for 8 h) and SHH (100 ng/ml for 6 h). The whole-cell lysate was subjected to pulldown with His beads. All panels are representative results from three or more independent experiments