Sarah Kit Leng Lui et al USP26 stabilizes SM7 MO reports xpanded View igures igure V1. USP26 enhances SM2 phosphorylation and T-b-mediated transcription. raph representing relative luciferase values obtained from U screen. 293T cells were transfected with -Luc and indicated U pools. orty-eight hours later, cells were treated with T-b for 16 h and a luciferase assay was performed. ata are mean S of triplicate samples. Table indicates relative luciferase value of each gene tested in (). Haat cells stably transduced with a hairpin targeting USP26 or vector control were stimulated with T-b for 3 h. PI1, KN1 (p21), T, LI, SM7, and USP26 mrn levels relative to PH or 18S are shown as evaluated by quantitative real-time PR. ata are mean S of triplicate samples. 293T cells were stably infected with two independent hairpins (L2 and L3) targeting USP26 and treated with T-b overnight. Whole-cell extracts were probed with the indicated antibodies. 293T cells were stably infected with three independent hairpins (L1, L2, and L3) targeting USP26. USP26 mrn levels relative to 18S are shown as evaluated by quantitative real-time PR. ata are mean S of triplicate samples. 293T cells transfected with sirn targeting USP26 or control vector were treated with T-b overnight. Whole-cell extracts were probed with the indicated antibodies. 293T cells expressing knockdown vectors targeting USP26 or control vector were treated with T-b overnight. Whole-cell extracts were probed with the indicated antibodies. ª 2017 The uthors MO reports V1
MO reports USP26 stabilizes SM7 Sarah Kit Leng Lui et al H igure V2. USP26 binds to and deubiquitinates SM7. 293T cells were transfected as indicated with P-USP26 and lag-tagged SM3 and SM7. fter 48 h, cells were lysed and immunoprecipitated with anti-lag affinity resin. Whole-cell extracts were probed with the indicated antibodies. 293T cells were transfected with P-USP26, and cells were lysed and immunoprecipitated with anti-p affinity resin. Whole-cell extracts were probed for SM3. 293T cells were transfected as indicated with P-USP26 and lag-tagged SM7. fter 48 h, cells were treated with T-b for 1 h and lysed. lag-tagged proteins were immunoprecipitated with anti-lag affinity resin and whole-cell extracts were probed with the indicated antibodies. 293T cells were transfected as indicated with P-USP26 and lag-tagged SM6. fter 48 h, cells were treated with T-b and lysed. lag-tagged proteins were immunoprecipitated with anti-lag affinity resin and whole-cell extracts were probed with the indicated antibodies. 293T cells transfected with L-SM7, P-USP26, control vector, and H-tagged ubiquitin. ollowing immunoprecipitation of SM7, lysates were resolved by SS P and probed with the indicated antibodies. ndogenous ubiquitination assay. 293T cells transfected with L-SM7, P-USP26, or control vector. ollowing immunoprecipitation of SM7, lysates were resolved by SS P and probed with the indicated antibodies. 293T cells were transfected as indicated with P-USP26, lag-tagged SM7, and Myc-tagged SMUR2. fter 48 h, cells were lysed and immunoprecipitated with anti-lag affinity resin overnight, eluted with anti-lag peptide, and re-immunoprecipitated with anti-myc affinity resin. Whole-cell extracts were probed with the indicated antibodies. H 293T cells were transfected as indicated with P-USP26, H-tagged SM7, and Myc-tagged SMUR1. fter 48 h, cells were lysed and immunoprecipitated with anti-lag affinity resin overnight, cleaved with an anti-lag peptide, and re-immunoprecipitated with anti-myc affinity resin. Whole-cell extracts were probed with the indicated antibodies. V2 MO reports ª 2017 The uthors
Sarah Kit Leng Lui et al USP26 stabilizes SM7 MO reports igure V3. Regulation of the T-b by USP26 in breast cancer and M cell lines. M7 cells transfected with sirn targeting USP26 or control vector were treated with T-b overnight. Whole-cell extracts were probed with the indicated antibodies (top panel). orresponding USP26 mrn levels relative to 18S are shown as evaluated by quantitative real-time PR (bottom panel). ata are mean S of triplicate samples. M-M-231 (), U373 (), and PT () cells stably expressing lentiviral knockdown vectors targeting USP26 or control vector were treated with T-b overnight. Whole-cell extracts were probed with the indicated antibodies (top panel). orresponding USP26 mrn levels relative to 18S are shown as evaluated by quantitative real-time PR (bottom panel). ata are mean S of triplicate samples. PT cells stably transduced with a hairpin targeting USP26 or vector control were stimulated with T-b for 3 h. T, LI, and SM7 mrn levels relative to 18S are shown as evaluated by quantitative real-time PR. ata are mean S of triplicate samples. M-M-231 cells stably expressing lentiviral knockdown vectors targeting USP26 or control vector were treated with transfected with L-SM7 (1 lg). Wholecell extracts were probed with the indicated antibodies. * denotes background band. U373 cells stably expressing lentiviral knockdown vectors targeting USP26 or control vector were treated with transfected with L-SM7 (1 lg). Whole-cell extracts were probed with the indicated antibodies. * denotes background band. ª 2017 The uthors MO reports V3
MO reports USP26 stabilizes SM7 Sarah Kit Leng Lui et al igure V4. xpression of SM7 and USP26 following T-b induction in breast cancer and M cell lines. The breast cancer cell lines M7 (), M-M-231 (), T47 (), and L51 () were stimulated with T-b for 1 and 3 h. USP26 mrn (left panel) and SM7 (right panel) levels relative to 18S are shown as evaluated by quantitative real-time PR. ata are mean S of triplicate samples. The glioblastoma cancer cell lines U373 (), PT (), and 172 () were stimulated with T-b for 1 and 3 h. USP26 mrn (left panel) and SM7 (right panel) levels relative to 18S are shown as evaluated by quantitative real-time PR. ata are mean S of triplicate samples. V4 MO reports ª 2017 The uthors
Sarah Kit Leng Lui et al USP26 stabilizes SM7 MO reports igure V5. orrelation of T-b pathway components with overall survival in M. Validation of the USP26 antibody for immunohistochemistry. 293T cells stably infected with knockdown vectors targeting USP26 were analyzed by immunohistochemistry for USP26 expression. Representative images are shown. To prepare sections, cell pellets were fixed in formalin and embedded in paraffin. Red staining indicates positive immunoreactivity. Scale bars: 50 lm. Kaplan Meier curves of glioblastoma patients (n = 329) with TRI (), TRII (), TRIII (), and SM7 () (RMRNT). P-value was obtained by log-rank test. ª 2017 The uthors MO reports V5