rabbit polyclonal npepps antibody ta308014  (OriGene)

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) The heatmap represents LC-MS/MS analysis of NPEPPS FLAG pull-down using protein lysates from BCa cell lines (KU1919 and T24; n = 3 independent lysates per cell line). The relative strength of interaction is scaled from 0 to 1. Genes annotated as playing a role in platinum drug resistance by Huang et al. are highlighted. Results from a CRISPR screen reported in Jones et al. identifying synthetic gene-to-drug interactions against cisplatin-based chemotherapy in treatment-resistant cell lines are shown. *FDR < 0.05. ( B ) Proteins identified in the NPEPPS FLAG screen as black dots and the VRAC subunits (LRRC8A-E) as red dots are mapped onto the CRISPR screen results. NPEPPS is highlighted in orange. ( C ) Affinity tag (FLAG), or IgG control, IP of protein lysates from NPEPPS FLAG in KU1919 and T24 cells was immunoblotted for NPEPPS, LRRC8A, and LRRC8D.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Liquid Chromatography with Mass Spectroscopy, CRISPR, Control

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Untargeted metabolomics in KU1919 cells with shRNA-mediated NPEPPS suppression or control shRNA. Taurine levels are reported in control (PBS) and cisplatin (Cis) treatment conditions. Targeted metabolomic measured levels of taurine are reported in T24 cells in control (PBS) and cisplatin (Cis) treatment conditions. hr, hours. ( B ) CyTOF in KU1919 cells shows intracellular cisplatin levels after 4 hours of 10 μM cisplatin with siRNA-mediated suppression of VRAC subunits LRRC8A-E compared to control (scramble) siRNA. Median intracellular cisplatin measurements across biological triplicates were normalized to the siRNA control and compared using a one-way ANOVA (** P < 0.01; ****P < 0.001). ( C ) Intracellular cisplatin levels for KU1919, T24, and 5637 cells with siRNA-mediated knockdown of NPEPPS alone, LRRC8A alone, or the combination of NPEPPS and LRRC8A knockdown. All samples were normalized to siRNA control samples and compared using a one-way ANOVA (* P < 0.05; ** P < 0.01; **** P < 0.0001). n.s., not significant. Immunoblot validation of the knockdowns is reported with native NPEPPS and LRRC8A antibodies. ( D ) KU1919, 5637, and T24 cells made resistant to GemCis were treated with cisplatin (10 μM) or PBS for 48 hours. Immunoblots with NPEPPS, LRRC8A, and phospho-γ-H2AX antibodies are shown, comparing shRNA-mediated knockdown of NPEPPS to shRNA scramble controls.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: shRNA, Control, Knockdown, Western Blot, Biomarker Discovery

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Confocal microscopy images illustrating the cellular localization of NPEPPS and LRRC8A via NPEPPS FLAG or LRRC8A FLAG in both KU1919 and T24 cells. Shown with and without differential interference contrast (DIC). Scale bars, 20 μm. ( B ) Immunoblots for NPEPPS and LRRC8A after FLAG affinity pull-down from NPEPPS FLAG in cytosolic and membranal fractions of T24 cell lysates. ( C ) Dual staining of LRRC8A and NPEPPS in LRRC8A FLAG KU1919 and T24 cells using anti-FLAG primary and Alexa Fluor (AF) 594 secondary antibodies for LRRC8A and anti-NPEPPS primary and AF647 secondary antibodies for NPEPPS. Scale bars, 20 μm [for nonzoomed images (top row)] and 2 μm [for zoomed images (bottom row)]. ( D ) Confocal microscopy showing colocalization of NPEPPS and LRRC8A in LRRC8A-mCherry reporter-containing T24 cells stained with anti-NPEPPS primary and AF647 secondary antibodies. Scale bars, 20 μm [for nonzoomed images (top row)] and 2 μm [for zoomed images (bottom row)]. NPEPPS-LRRC8A colocalization was calculated in ImageJ by PCC (r or Correlation), and statistical significance was evaluated by unpaired t test.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Confocal Microscopy, Western Blot, Staining

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Quantification of NPEPPS enzymatic activity on the H-Leu-AMC substrate with vehicle (PBS, 0 μM) or tosedostat (20 μM) treatment in NPEPPS FLAG KU1919 parental cells. a.u., arbitrary units. ( B ) Immunoblot of NPEPPS and LRRC8A in KU1919 parental and GemCis-resistant cells after IP with native NPEPPS or LRRCA antibodies following 72 hours of PBS control (0 μM) or tosedostat (10 μM) treatment. ( C ) Quantification of NPEPPS-LRRC8A colocalization by FRET-AB in LRRC8A FLAG KU1919 parental cells with vehicle (PBS, 0 μM) or tosedostat (20 μM) treatment. The relative quantification is reported as FRET efficiency (%) and statistical comparisons were made using the Mann-Whitney U test. ( D ) Immunofluorescence confocal microscopy of NPEPPS FLAG constructs [NPEPPS −/−(WT-FLAG) and NPEPPS −/−(E353V-FLAG) ] in cisplatin-resistant KU1919 cells. Scale bars, 20 μm. ( E ) Quantification of NPEPPS enzymatic activity on the H-Leu-AMC substrate in KU1919-Cis cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . ( F ) Immunoblots of NPEPPS and LRRC8A following anti-FLAG IP in parental and cisplatin-resistant KU1919 cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . ( G ) Intracellular cisplatin concentrations were measured by CyTOF in triplicate experiments in WT KU1919-Cis cells or KU1919-Cis cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . Cisplatin concentrations were normalized to the unmodified cisplatin-resistant KU1919 cells, and comparisons were made using one-way ANOVA (**** P < 0.0001). ( H ) Cisplatin IC 50 was measured in technical and biological triplicate by IncuCyte Zoom analysis over 120 hours of treatment in WT KU1919-Cis cells or KU1919-Cis cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . Results are reported as the mean IC 50 ± SD. Comparisons were made using one-way ANOVA (* P < 0.05).

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Activity Assay, Western Blot, Control, Quantitative Proteomics, MANN-WHITNEY, Immunofluorescence, Confocal Microscopy, Construct, Expressing

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Mice were injected with 4 × 10 6 cells in each flank to generate tumors using KU1919-Cis cells with NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) expression. When engrafted tumors reached 100 mm 3 , mice were randomized to control or treatment groups and received either cisplatin (2 mg/kg; by intraperitoneal injection three times per week) or PBS control (equal volume of saline by intraperitoneal injection three times per week). Tumor size was measured with calipers every 2 to 3 days throughout the duration of treatment. Differences in growth rates by tumor genotype were evaluated by a two-way ANOVA mixed effects model. ( B ) Wet tumor weights were measured for each group. Comparisons were made using a one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). ( C ) Fluorescence-based enzyme assay to quantify the catalytic activity of NPEPPS across the cells isolated from mouse tumors using H-Leu-AMC as the substrate. ( D ) DNA damage was measured by flow cytometry–based quantification of phosphorylated histone H2AX. Comparisons were made using a one-way ANOVA (* P < 0.05; *** P < 0.001). ( E ) Stratification of patient survival by above-median or below-median expression of the ratio of NPEPPS /Avg( LRRC8A-E ) in BCa tumor samples before cisplatin treatment. Analysis of survival differences was conducted in R, and statistical significance was evaluated by Cox proportional hazard ratios and the log-rank test. Time to median survival is indicated by a dashed line. ( F ) Patient-derived organoids were evaluated for mRNA expression and cisplatin IC 50 . Left: Average counts per million mRNA expression of VRAC subunits LRRC8A-E and NPEPPS in each patient-derived organoid. Right: The correlation of NPEPPS /Avg( LRRC8A-E ) expression with cisplatin sensitivity is plotted ( r 2 = 0.7172). Organoids from patient 1 were derived from a cystectomy sample, and the patient was not given neoadjuvant chemotherapy (NAC). Organoids from patients 2 to 5 were derived from the transurethral resection of bladder tumor samples before the patients received NAC.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Injection, Expressing, Control, Saline, Fluorescence, Enzymatic Assay, Activity Assay, Isolation, Flow Cytometry, Derivative Assay

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Immunoblots of NPEPPS in cisplatin-resistant Caov-3 cells (Caov-3-Cis) with WT NPEPPS (Caov-3-Cis-gCtrl) or KO of NPEPPS (Caov-3-Cis-gNPEPPS −/− ). Intracellular cisplatin concentrations were measured by CyTOF in triplicate experiments in WT, chemotherapy-sensitive Caov-3-Par cells, WT, chemotherapy-resistant Caov-3-Cis cells, or engineered Caov-3-Cis cells with control (-gCtrl) or NPEPPS KO (- NPEPPS −/− ). Cisplatin concentrations were normalized to unmodified cisplatin-sensitive Caov-3-Par cells, and comparisons were made using one-way ANOVA (* P < 0.05; ** P < 0.01). Cisplatin IC was measured in technical and biological triplicate in WT Caov-3-Par, Cis, Cis-gCtrl, or Cis-gNPEPPS −/− cells. Results are reported as the mean IC ± SD. Comparisons were made using one-way ANOVA (**** P < 0.0001). ( B ) Immunoblots of NPEPPS in WT HEK293T cells, HEK293T-gCtrl cells, or HEK293T-gNPEPPS −/− cells. Intracellular cisplatin concentrations were measured by CyTOF, and comparisons were made using one-way ANOVA (**** P < 0.0001). Cisplatin IC was measured in technical and biological triplicate and reported as the mean IC ± SD. Comparisons were made using one-way ANOVA (* P < 0.05; ** P < 0.01). ( C ) GE data from paired cisplatin-sensitive and cisplatin-resistant cancer cell lines representing colorectal, kidney, breast, lung , and ovarian cancers were evaluated for the expression ratio of NPEPPS /Avg( LRRC8A-E ). The comparison was made using a paired t test. ( D ) Synthetic lethal and synthetic resistant z scores of NPEPPS and LRRC8A-E across three cisplatin datasets were evaluated by CRISPR screening in the RPE1 cell line and retrieved from the Olivieri et al. CRISPR screen repository . ( E ) Stratification of patient survival by above-median or below-median expression of the ratio of NPEPPS /Avg( LRRC8A-E ) in tumor samples from HGSC [Yoshihara et al. ] and CESC [TCGA ] prior to platinum-based treatment. Time to median survival is indicated by a dashed line (omitted where survival is >50% at all time points), and comparisons were made using the log-rank test.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Western Blot, Control, Expressing, Comparison, CRISPR

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) The heatmap represents LC-MS/MS analysis of NPEPPS FLAG pull-down using protein lysates from BCa cell lines (KU1919 and T24; n = 3 independent lysates per cell line). The relative strength of interaction is scaled from 0 to 1. Genes annotated as playing a role in platinum drug resistance by Huang et al. are highlighted. Results from a CRISPR screen reported in Jones et al. identifying synthetic gene-to-drug interactions against cisplatin-based chemotherapy in treatment-resistant cell lines are shown. *FDR < 0.05. ( B ) Proteins identified in the NPEPPS FLAG screen as black dots and the VRAC subunits (LRRC8A-E) as red dots are mapped onto the CRISPR screen results. NPEPPS is highlighted in orange. ( C ) Affinity tag (FLAG), or IgG control, IP of protein lysates from NPEPPS FLAG in KU1919 and T24 cells was immunoblotted for NPEPPS, LRRC8A, and LRRC8D.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Liquid Chromatography with Mass Spectroscopy, CRISPR, Control

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Untargeted metabolomics in KU1919 cells with shRNA-mediated NPEPPS suppression or control shRNA. Taurine levels are reported in control (PBS) and cisplatin (Cis) treatment conditions. Targeted metabolomic measured levels of taurine are reported in T24 cells in control (PBS) and cisplatin (Cis) treatment conditions. hr, hours. ( B ) CyTOF in KU1919 cells shows intracellular cisplatin levels after 4 hours of 10 μM cisplatin with siRNA-mediated suppression of VRAC subunits LRRC8A-E compared to control (scramble) siRNA. Median intracellular cisplatin measurements across biological triplicates were normalized to the siRNA control and compared using a one-way ANOVA (** P < 0.01; ****P < 0.001). ( C ) Intracellular cisplatin levels for KU1919, T24, and 5637 cells with siRNA-mediated knockdown of NPEPPS alone, LRRC8A alone, or the combination of NPEPPS and LRRC8A knockdown. All samples were normalized to siRNA control samples and compared using a one-way ANOVA (* P < 0.05; ** P < 0.01; **** P < 0.0001). n.s., not significant. Immunoblot validation of the knockdowns is reported with native NPEPPS and LRRC8A antibodies. ( D ) KU1919, 5637, and T24 cells made resistant to GemCis were treated with cisplatin (10 μM) or PBS for 48 hours. Immunoblots with NPEPPS, LRRC8A, and phospho-γ-H2AX antibodies are shown, comparing shRNA-mediated knockdown of NPEPPS to shRNA scramble controls.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: shRNA, Control, Knockdown, Western Blot, Biomarker Discovery

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Confocal microscopy images illustrating the cellular localization of NPEPPS and LRRC8A via NPEPPS FLAG or LRRC8A FLAG in both KU1919 and T24 cells. Shown with and without differential interference contrast (DIC). Scale bars, 20 μm. ( B ) Immunoblots for NPEPPS and LRRC8A after FLAG affinity pull-down from NPEPPS FLAG in cytosolic and membranal fractions of T24 cell lysates. ( C ) Dual staining of LRRC8A and NPEPPS in LRRC8A FLAG KU1919 and T24 cells using anti-FLAG primary and Alexa Fluor (AF) 594 secondary antibodies for LRRC8A and anti-NPEPPS primary and AF647 secondary antibodies for NPEPPS. Scale bars, 20 μm [for nonzoomed images (top row)] and 2 μm [for zoomed images (bottom row)]. ( D ) Confocal microscopy showing colocalization of NPEPPS and LRRC8A in LRRC8A-mCherry reporter-containing T24 cells stained with anti-NPEPPS primary and AF647 secondary antibodies. Scale bars, 20 μm [for nonzoomed images (top row)] and 2 μm [for zoomed images (bottom row)]. NPEPPS-LRRC8A colocalization was calculated in ImageJ by PCC (r or Correlation), and statistical significance was evaluated by unpaired t test.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Confocal Microscopy, Western Blot, Staining

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Quantification of NPEPPS enzymatic activity on the H-Leu-AMC substrate with vehicle (PBS, 0 μM) or tosedostat (20 μM) treatment in NPEPPS FLAG KU1919 parental cells. a.u., arbitrary units. ( B ) Immunoblot of NPEPPS and LRRC8A in KU1919 parental and GemCis-resistant cells after IP with native NPEPPS or LRRCA antibodies following 72 hours of PBS control (0 μM) or tosedostat (10 μM) treatment. ( C ) Quantification of NPEPPS-LRRC8A colocalization by FRET-AB in LRRC8A FLAG KU1919 parental cells with vehicle (PBS, 0 μM) or tosedostat (20 μM) treatment. The relative quantification is reported as FRET efficiency (%) and statistical comparisons were made using the Mann-Whitney U test. ( D ) Immunofluorescence confocal microscopy of NPEPPS FLAG constructs [NPEPPS −/−(WT-FLAG) and NPEPPS −/−(E353V-FLAG) ] in cisplatin-resistant KU1919 cells. Scale bars, 20 μm. ( E ) Quantification of NPEPPS enzymatic activity on the H-Leu-AMC substrate in KU1919-Cis cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . ( F ) Immunoblots of NPEPPS and LRRC8A following anti-FLAG IP in parental and cisplatin-resistant KU1919 cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . ( G ) Intracellular cisplatin concentrations were measured by CyTOF in triplicate experiments in WT KU1919-Cis cells or KU1919-Cis cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . Cisplatin concentrations were normalized to the unmodified cisplatin-resistant KU1919 cells, and comparisons were made using one-way ANOVA (**** P < 0.0001). ( H ) Cisplatin IC 50 was measured in technical and biological triplicate by IncuCyte Zoom analysis over 120 hours of treatment in WT KU1919-Cis cells or KU1919-Cis cells expressing NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) . Results are reported as the mean IC 50 ± SD. Comparisons were made using one-way ANOVA (* P < 0.05).

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Activity Assay, Western Blot, Control, Quantitative Proteomics, MANN-WHITNEY, Immunofluorescence, Confocal Microscopy, Construct, Expressing

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Mice were injected with 4 × 10 6 cells in each flank to generate tumors using KU1919-Cis cells with NPEPPS −/−(WT-FLAG) or NPEPPS −/−(E353V-FLAG) expression. When engrafted tumors reached 100 mm 3 , mice were randomized to control or treatment groups and received either cisplatin (2 mg/kg; by intraperitoneal injection three times per week) or PBS control (equal volume of saline by intraperitoneal injection three times per week). Tumor size was measured with calipers every 2 to 3 days throughout the duration of treatment. Differences in growth rates by tumor genotype were evaluated by a two-way ANOVA mixed effects model. ( B ) Wet tumor weights were measured for each group. Comparisons were made using a one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). ( C ) Fluorescence-based enzyme assay to quantify the catalytic activity of NPEPPS across the cells isolated from mouse tumors using H-Leu-AMC as the substrate. ( D ) DNA damage was measured by flow cytometry–based quantification of phosphorylated histone H2AX. Comparisons were made using a one-way ANOVA (* P < 0.05; *** P < 0.001). ( E ) Stratification of patient survival by above-median or below-median expression of the ratio of NPEPPS /Avg( LRRC8A-E ) in BCa tumor samples before cisplatin treatment. Analysis of survival differences was conducted in R, and statistical significance was evaluated by Cox proportional hazard ratios and the log-rank test. Time to median survival is indicated by a dashed line. ( F ) Patient-derived organoids were evaluated for mRNA expression and cisplatin IC 50 . Left: Average counts per million mRNA expression of VRAC subunits LRRC8A-E and NPEPPS in each patient-derived organoid. Right: The correlation of NPEPPS /Avg( LRRC8A-E ) expression with cisplatin sensitivity is plotted ( r 2 = 0.7172). Organoids from patient 1 were derived from a cystectomy sample, and the patient was not given neoadjuvant chemotherapy (NAC). Organoids from patients 2 to 5 were derived from the transurethral resection of bladder tumor samples before the patients received NAC.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Injection, Expressing, Control, Saline, Fluorescence, Enzymatic Assay, Activity Assay, Isolation, Flow Cytometry, Derivative Assay

Journal: Science Advances

Article Title: Regulation of volume-regulated anion channels alters sensitivity to platinum chemotherapy

doi: 10.1126/sciadv.adr9364

Figure Lengend Snippet: ( A ) Immunoblots of NPEPPS in cisplatin-resistant Caov-3 cells (Caov-3-Cis) with WT NPEPPS (Caov-3-Cis-gCtrl) or KO of NPEPPS (Caov-3-Cis-gNPEPPS −/− ). Intracellular cisplatin concentrations were measured by CyTOF in triplicate experiments in WT, chemotherapy-sensitive Caov-3-Par cells, WT, chemotherapy-resistant Caov-3-Cis cells, or engineered Caov-3-Cis cells with control (-gCtrl) or NPEPPS KO (- NPEPPS −/− ). Cisplatin concentrations were normalized to unmodified cisplatin-sensitive Caov-3-Par cells, and comparisons were made using one-way ANOVA (* P < 0.05; ** P < 0.01). Cisplatin IC was measured in technical and biological triplicate in WT Caov-3-Par, Cis, Cis-gCtrl, or Cis-gNPEPPS −/− cells. Results are reported as the mean IC ± SD. Comparisons were made using one-way ANOVA (**** P < 0.0001). ( B ) Immunoblots of NPEPPS in WT HEK293T cells, HEK293T-gCtrl cells, or HEK293T-gNPEPPS −/− cells. Intracellular cisplatin concentrations were measured by CyTOF, and comparisons were made using one-way ANOVA (**** P < 0.0001). Cisplatin IC was measured in technical and biological triplicate and reported as the mean IC ± SD. Comparisons were made using one-way ANOVA (* P < 0.05; ** P < 0.01). ( C ) GE data from paired cisplatin-sensitive and cisplatin-resistant cancer cell lines representing colorectal, kidney, breast, lung , and ovarian cancers were evaluated for the expression ratio of NPEPPS /Avg( LRRC8A-E ). The comparison was made using a paired t test. ( D ) Synthetic lethal and synthetic resistant z scores of NPEPPS and LRRC8A-E across three cisplatin datasets were evaluated by CRISPR screening in the RPE1 cell line and retrieved from the Olivieri et al. CRISPR screen repository . ( E ) Stratification of patient survival by above-median or below-median expression of the ratio of NPEPPS /Avg( LRRC8A-E ) in tumor samples from HGSC [Yoshihara et al. ] and CESC [TCGA ] prior to platinum-based treatment. Time to median survival is indicated by a dashed line (omitted where survival is >50% at all time points), and comparisons were made using the log-rank test.

Article Snippet: NPEPPS and LRRC8A have been probed using the rabbit polyclonal NPEPPS antibody (1:1000; Origene, TA308014), rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio, LS-C290818 and LS-B16989), and rabbit IgG polyclonal LRRC8D antibody (1:1000, Sino Biological, 104245-T32).

Techniques: Western Blot, Control, Expressing, Comparison, CRISPR

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) Differential gene expression of the 46 common synthetic lethal genes as measured by RNAseq across all cell lines, comparing the treatment resistant derivative (Gem-, Cis-, GemCis-resistant) to the associated parental cell line. Asterisks indicate a statistically significant result (moderated t-test, *FDR < 0.05). The bar plot on top is the aggregate count of significant results across all 15 comparisons. Genes are ranked by the count of statistically significant upregulated hits. ( B ) RNAseq (compared to parentals; *FDR < 0.05), ( C ) mass spectrometry proteomics (compared to parentals, *FDR < 0.25), and ( D ) CRISPR screen results for NEPPSP (*FDR < 0.05). ( E ) Representative immunoblots and densitometry quantification for independent triplicates (mean ± SEM) for NPEPPS in all cell lines (*FDR < 0.05).

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: Gene Expression, Mass Spectrometry, CRISPR, Western Blot

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) NPEPPS was found to be synthetic lethal with cisplatin in a CRISPR screen for 27 genotoxic agents in RPE1 cells by . ( B ) Immunoblot for NPEPPS across several control and shRNAs targeting NPEPPS. ( C, D ) KU1919-GemCis cells with knockdown of NPEPPS treated with increasing doses of cisplatin or gemcitabine. A total of 3 technical replicates per dose (mean ± SEM). Independent experiments are reported in Figure S5 . ( E ) NPEPPS mRNA is upregulated in response to cisplatin treatment in a dose dependent manner in both KU1919-Par and KU1919-GemCis cells. Independent triplicate experiments are shown (mean ± SEM) (t-test compared to 0μM; *p < 0.05, **p < 0.05). ( F ) Pharmacologic targeting of NPEPPS with tosedostat in GemCis-resistant cells treated with cisplatin, gemcitabine, or gemcitabine plus cisplatin. A total of 3 technical replicates per dose are shown (mean ± SEM). Independent experiments are reported in Figure S6 .

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: CRISPR, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) NPEPPS is found to interact with all VRAC subunits, LRRC8A-E, as reported in the BioPlex interactome . ( B ) Anti-FLAG was used against KU1919 and T24 parental cell lines as controls and overexpressing FLAG tagged NPEPPS, KU1919 and T24 cells. The immunoprecipitant was immunoblotted for NPEPPS, LRRC8A, and LRRC8D, demonstrating that LRRC8A and LRRC8D are pulled down in complex with NPEPPS. ( C ) Genes ranked based on log2 fold change from the synthetic lethal CRISPR screens across all cell lines. LRRC8A-E and the 46 common synthetic lethal genes are labeled. ( D, E ) Knockout of LRRC8A and LRRC8D through the CRISPR screen resulted in increased cell growth upon gemcitabine plus cisplatin treatment in GemCis- resistant cell lines (moderated t-test; *FDR < 0.05). ( F, G ) LRRC8A and LRRC8D gene expression measured by RNAseq (compared to parentals; *FDR < 0.05).

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: CRISPR, Labeling, Knock-Out, Gene Expression

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: ( A ) Volcano plot of metabolites measured from KU1919-GemCis cells with or without NPEPPS knockdown (shN39). Time and treatment (cisplatin 10μM) were covariates in the linear model to calculate differential expression using a moderated t-test; horizontal grey line is -log10(FDR = 0.05). ( B )Taurine abundance measured in KU1919-GemCis cells with non- targeting shRNA controls or shRNA targeting NPEPPS (shN39). Cells were also measured at 48 hours treated with 10μM cisplatin or PBS. ( C ) Intracellular cisplatin levels in ( C ) KU1919 or ( D ) 5637 cells were measured after 4 hours of 10μM cisplatin treatment using CyTOF, with the number of cells analyzed as indicated. Immunoblot of LRRC8A, LRRC8D, and γH2AX in ( E ) KU1919-GemCis-shCtrl1 and KU1919- GemCis-shN39 or ( F ) 5637-GemCis-shCtrl1 and 5637-GemCis-shN39 cells treated with PBS or 10μM cisplatin for 48 hours. ( G ) Intracellular cisplatin concentrations were measured for KU1919 parental and then for untargeted knockdown (siCtrl) and targeted knockdown of NPEPPS (siNPEPPS), LRRC8A (siLRRC8A), and the combination of NPEPPS and LRRC8A (siNPEPPS+siLRRC8A). ( H ) Tumor volume of KU1919-GemCis xenografts measured over time and across 4 treatment groups considering non- targeting shRNA controls (shCtrl1), shRNA targeting NPEPPS (shN39), PBS vehicle control (PBS), or gemcitabine plus cisplatin treatment (GemCis). ( I ) Survival analysis of xenograft models with a defined endpoint of a tumor volume > 2cm 3 . Logrank test was applied to test significance. ( J ) Survival analysis of muscle-invasive bladder cancer in the TCGA stratified based on copy number amplification, gain or overexpression of LRRC8A or LRRC8D. Patients all had a record of cisplatin-based chemotherapy treatment. ( K ) Survival analysis for patients stratified by LRRC8A or LRRC8D as in ( J ), but that did not have any record of cisplatin-based treatments.

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: Knockdown, Quantitative Proteomics, shRNA, Western Blot, Control, Amplification, Over Expression

Journal: bioRxiv

Article Title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels

doi: 10.1101/2021.03.04.433676

Figure Lengend Snippet: Normal functioning cells will import cisplatin through the volume regulated anion channels (VRAC), with LRRC8A and LRRC8D being the primary subunits. A mechanism of cisplatin resistance is to inherently down-regulate VRACs. We propose that NPEPPS interacts with LRRC8A or LRRC8D directly to decrease VRAC activity, which prevents export of taurine and import of cisplatin, hence driving cisplatin resistance.

Article Snippet: NPEPPS and LRRC8A has been probed using Rabbit polyclonal NPEPPS antibody (1:1000; Origene), Rabbit IgG polyclonal LRRC8A antibody (1:1000, LSBio) and Rabbit IgG polyclonal LRRC8D antibody (1:1000, SinoBiological).

Techniques: Activity Assay