human recombinant c terminal flag tag  (BPS Bioscience)

Journal: Nature communications

Article Title: MICU1 drives glycolysis and chemoresistance in ovarian cancer.

doi: 10.1038/ncomms14634

Figure Lengend Snippet: Figure 4 | MICU1-mediated Ca2 þ buffering in OvCa cells evaluated using cisplatin. (a) CP20 cells loaded with the fluorescent dyes Rhod-2AM (5 mM), 10 mM cisplatin caused rapid increase in [Ca2 þ]m. (b,c) Silencing of MICU1 by siRNA increased [Ca2 þ]m responses to cisplatin in CP20 (B) or OV90 (c) cells loaded with Rhod-2AM (5 mM) and values are mean±s.d. (d) Normalized GCaMP2-mt fluorescence in shRNA transfected (shCTL-OV90) or MICU1- specific shRNA transfected (shMICU1-OV90) cells stimulated with cisplatin (10 mM). The experiment was repeated three times (total no. of cells cell quantified: shCTL-OV90 n ¼ 158 and shMICU1-OV90 n ¼ 128). (e) Changes in [Ca2 þ]m in OSE cells expressing Flag-MICU1 upon 10 mM cisplatin treatment as compared to empty vector (EV) transfected cells and values are mean±s.d. (f) Normalized GCaMP2-mt fluorescence in FTE188 cells transfected with empty vector (FTE þ EV) or Flag-MICU1 (FTE þ Flag-MICU1) cells stimulated with cisplatin (10 mM). The experiment was repeated three times (total no. of cells quantified: FTE þ EV n ¼ 135 and FTE þ Flag MICU1 n ¼ 433). (g) Cisplatin causes similar release of [Ca2 þ]ER in presence or absence of MICU1 as determined in OV90 cells loaded with [Ca2 þ]ER indicator dye Fluo-5N (5 mM) and values are mean±s.d. (h) Disruption of [Ca2 þ]ER release by Pyr6 (50 nM) prevents cisplatin-induced changes in [Ca2 þ]m of OV90 cells. Cells were pretreated with Pyr6 (50 nM) and Rhod-2AM (5 mM) for 15 min followed by 10 mM cisplatin treatment, fluorescence for Rhod-2AM was determined. Data are expressed as mean±s.d. *Po0.05 for comparisons among more than two groups, we performed ANOVA with Bonferroni’s correction. Po0.05 was considered statistically significant. All tests were two-sided. Horizontal line represents statistical comparison between respective groups. All the experiments were repeated independently at least three times and in triplicate.

Article Snippet: Transfection of OSE and FTE188 cells with Flag-MICU1 (pLYS1-MICU1-Flag was a gift from Vamsi Mootha, Addgene plasmid # 50058) (ref. 73) or Empty vector was performed using Lipofectamine 3000 with 4 mg plasmid DNA.

Techniques: shRNA, Transfection, Expressing, Plasmid Preparation, Disruption, Comparison

Journal: Nature communications

Article Title: MICU1 drives glycolysis and chemoresistance in ovarian cancer.

doi: 10.1038/ncomms14634

Figure Lengend Snippet: Figure 5 | MICU1 negatively regulates OxPhos function and induces lactate production. (a,b) Oxygen consumption rate (OCR) was measured using the Seahorse XF-96 analyser in OSE (a) and FTE188 cells expressing EV (empty vector) or FLAG-MICU1. An XF trace representing mean±s.e.m. of at least three technical replicates per group is shown. Cells were sequentially treated with 2 mM Oligomycin (O), 0.5 mM FCCP (F) and 1 mM each of Antimycin þ Rotenone (A þ R) at indicated time points. (c) FCCP stimulated response were derived from the XF trace as in a,b from two independent experiments and represented as mean±s.e.m. (d) CP20 and (e) OV90 cells were transfected with scrambled siRNA (siCTL) or MICU1 siRNA and the oxygen consumption rate (OCR) measured using the Seahorse XF analyser. An XF trace representing mean±s.e.m. from at least three technical replicates per group is shown. (f) FCCP stimulated response were derived from the XF trace of two independent experiments and represented as mean±s.e.m. (g,h) Intracellular lactate was measured in MICU1 silenced OV90 (g) and CP20 (h) cells with treatment with cisplatin 10 mM or vehicle and values represent mean±s.d. (i) Intracellular lactate measurement in MICU1 overexpressing OSE and FTE188 cells as in g. *Po0.05 statistically significant when compared to EV (c,i) siCTL (f–h). Comparisons between two groups were evaluated using two-sided Student’s t-test with unequal variances. Horizontal bar represents statistical comparison between respective groups. All the experiments were repeated independently at least three times and in triplicates.

Article Snippet: Transfection of OSE and FTE188 cells with Flag-MICU1 (pLYS1-MICU1-Flag was a gift from Vamsi Mootha, Addgene plasmid # 50058) (ref. 73) or Empty vector was performed using Lipofectamine 3000 with 4 mg plasmid DNA.

Techniques: Expressing, Plasmid Preparation, Derivative Assay, Transfection, Comparison