Supplementary Figure from USP22 Interacts with PALB2 and Promotes Chemotherapy Resistance via Homologous Recombination of DNA Double-Strand Breaks

Supplementary Figure 1</p

Supplemental Figure Legend from USP22 Interacts with PALB2 and Promotes Chemotherapy Resistance via Homologous Recombination of DNA Double-Strand Breaks

Supplemental Figure Legend</p

Supplementary Figure S1 from c-Met Is a Potentially New Therapeutic Target for Treatment of Human Melanoma

Supplementary Figure S1 from c-Met Is a Potentially New Therapeutic Target for Treatment of Human Melanom

Results of screening PCR in two targeting GFF cells.

<p>A, PCR was performed using primers V01 and V02. The wild type allele was denoted by the 3-hK-ras^G12D-IRES-HSV1-tk allele was denoted by 6 kb fragment. B, PCR was performed using the primers V01 and V03. The wild type allele could not be denoted by any fragment using these pair of primers, and only the recombined LSL-hK-ras^G12D-IRES-HSV1-tk allele was denoted by 5.7 kb fragment. The fragments in lane1 and lane2 were purified from the gel and then were subject to sequencing. C, PCR was performed using primers V04 and V05. The wild allele type was denoted by the 8 kb fragment, and the recombined LSL-hK-ras^G12D-IRES-HSV1-tk allele was denoted by 10 kb fragment. WT: wild-type GFF cells. No. 1 and No. 2: Targeting cells. Position and orientation of PCR primers used for the analysis are depicted.</p

The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits EGF-induced phosphorylation of Src, PI3 kinase and STAT3 in human lung cancer cells.

<p><b>Panel A</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-phospho-Src (pY⁴¹⁶) (a), anti-phospho-p85/p55 PI3 kinase (pY⁴⁵⁸/pY¹⁹⁹) (b,c), anti-phospho-STAT3 (pY⁷⁰⁵) (d) and anti-actin (e) antibodies. <b>Panel B</b>: Graphical quantitation of immunoreactivity of experiments performed described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091577#pone-0091577-g003" target="_blank">Figure 3-B</a> and Panel A with normalization to total specific protein and n = 3 independent experiments per condition. An asterisk (*) indicates a statistically significant difference (p<0.05) from control. The error bars = standard deviation.</p

The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits EGF-induced recruitment/activation of the linker protein, Grb-2, and the scaffolding protein, Gab-1, in human lung cancer cells.

<p><b>Panel A</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX (1 hour pre-incubation), 10 ng/ml EGF for 15 minutes, or 100 nM MNTX and 10 ng/ml EGF. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates and immunoprecipitated material using anti-Grb-2 (a,c) and anti-EGFR (b,d) antibody. MNTX inhibits EGF-induced recruitment of Grb-2 to the EGFR. <b>Panel B</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY⁶²⁷ Gab-1 (a), anti-pY³⁰⁷ Gab-1 (b) and anti-actin (c) antibodies. MNTX attenuates EGF-induced Gab-1 tyrosine phosphorylation.</p

The mu opioid receptor (MOR) is recruited to the EGF receptor with EGF stimulation but does not regulate EGF receptor phosphorylation.

<p><b>Panel A</b>: Human H358 non-small cell lung cancer (NSCLC) cells were treated with no (control) or 100 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoprecipitated with anti-EGFR antibody. Immunoblots were performed on total cell lysates (left) and immunoprecipitated material (right) using anti-MOR (a) and anti-EGFR (b) antibodies. The mu opioid receptor is recruited to the EGFR with EGF stimulation. <b>Panel B</b>: Human H358 non-small cell lung cancer (NSCLC) cells were either untreated (control) or treated with 100 nM MNTX alone, 10 ng/ml EGF for 5, 15 or 30 minutes, or 100 nM MNTX and 10 ng/ml EGF for 5, 15, or 30 minutes. Cell lysates were obtained and immunoblotted using anti-pY⁸⁴⁵ EGFR (a), anti-pY⁹⁹² EGFR (b), anti-pY¹⁰⁴⁵ EGFR (c), anti-pY¹⁰⁶⁸ EGFR (d), anti-EGFR (e) and anti-actin (e) antibodies. MNTX does not inhibit EGF-induced EGFR tyrosine phosphorylation.</p

The peripheral mu opioid receptor antagonist, methylnaltrexone (MNTX), inhibits epidermal growth factor (EGF)-induced proliferation and migration of human lung cancer cells in a dose-dependent manner.

<p><b>Panel A</b>: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for methylnaltrexone (MNTX) inhibition of EGF-mediated proliferation using a MTS proliferation assay. Cells were growth in the presence of 100 ng/ml EGF and/or 0–250 nM MNTX for 72 hours. MNTX There is a statistically significant difference (p<0.05, indicated by an asterisks) between control and MNTX (10, 50, 100, 250 nM) treatment with n = 3 per condition and error bars = standard deviation. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091577#s2" target="_blank">Methods</a> section for experimental details. <b>Panel B</b>: Human H358 non-small cell lung cancer (NSCLC) cells were analyzed for methylnaltrexone (MNTX) inhibition of EGF-mediated migration using a transwell assay (8 uM pore size). Cells were allowed to migrate in the presence of 100 ng/ml EGF and/or 0–250 nM MNTX for 18 hours. There is a statistically significant difference (p<0.05, indicated by an asterisks) between control and MNTX (50, 100, 250 nM) treatment with n = 3 per condition and error bars = standard deviation. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091577#s2" target="_blank">Methods</a> section for experimental details.</p

Overexpression or silencing of the mu opioid receptor (MOR) or addition of opioids in human lung cancer cells regulates epithelial mesenchymal transition (EMT).

<p><b>Panel A</b>: Control (non-transfected)(C), stable vector control (VC) and MOR1 overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with EMT markers anti-vimentin (a), anti-Snail (b), anti-Slug (c), anti-claudin-1 (d), anti-ZO-1 (e), anti-MOR (f) and anti-actin (g) antibodies. An increase in vimentin, Snail and Slug expression and a decrease in claudin-1 and ZO-1 expression suggest an epithelial mesenchymal transition. <b>Panel B</b>: Graphical quantitation of immunoreactivity of experiments performed described in Panel A with n = 3 independent experiments per condition. An asterisk (*) indicates a statistically significant difference (p<0.05) from control with error bars = standard deviation. <b>Panel C</b>: H358 human NSCLC cells were either untreated, treated with 100 ng/ml epidermal growth factor (EGF), 100 nM DAMGO, morphine or fentanyl or 100 ng/ml insulin growth factor (IGF) for 96 hours, cell lysates obtained and immunoblotted with EMT markers anti-vimentin (a), anti-Snail (b), anti-Slug (c), anti-claudin-1 (d), anti-ZO-1 (e), anti-MOR (f) and anti-actin (g) antibodies. A decrease in vimentin, Snail and Slug expression and an increase in claudin-1 and ZO-1 expression suggest inhibition of epithelial mesenchymal transition. <b>Panel D</b>: Control shRNA or MOR shRNA H358 cell lines were generated, cell lysates obtained and immunoblotted with EMT markers anti-vimentin (a), anti-Snail (b), anti-Slug (c), anti-claudin-1 (d), anti-ZO-1 (e), anti-MOR (f) and anti-actin (g) antibodies. An increase in vimentin, Snail and Slug expression and a decrease in claudin-1 and ZO-1 expression suggest an epithelial mesenchymal transition.</p

Tumor formation in mice inoculated with Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells.

<p>A. Tumor growth curves of mice bearing Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells. Inset. Dissected tumor masses from the 5 mice. B. Coronal microPET images of nude mouse inoculated with Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells after injection of ¹⁸F-FDG. Red arrow indicates palpable tumor mass. Mouse inoculated with LSL-hK-ras^G12D-IRES-HSV1-tk GFF cells was used as a control.</p