Journal: PLOS One
Article Title: LSR overexpression induces chemoresistance in triple negative breast cancer cells through MDR1 upregulation and apoptosis attenuation
doi: 10.1371/journal.pone.0336124
Figure Lengend Snippet: A) The mRNA levels of MDR1 in 231/C and 231/LSR cells were determined by q -PCR analysis. B) The protein levels of MDR1 in 231/C and 231/LSR cells were detected with Western blot. C) LSR overexpression in MDA-MB-231 cells induces MDR1 promoter-mediated luciferase activity. 231/C and 231/LSR cells were co-transfected with plasmids encoding MDR1 promoter-firefly-luciferase and SV40 promoter-Renilla luciferase, followed by luciferase analysis after 48 hours. The relative MDR1-luciferase activity for each cell line was normalized to Renilla-luciferase signals. D & E) MDR1 inhibitor verapamil sensitized 231/LSR (D) and 231/C (E) cells to doxorubicin. The cells were treated with doxorubicin at indicated concentrations in the absence or presence of 10 μM verapamil for four days, followed by survival fraction detection with CCK-8 assays. The IC 50 of each group was calculated with GraphPad software. F) Inhibition of MDR1 enhances doxorubicin-induced apoptosis in 231/LSR cells measured by apoptosis ELISA. The cells were treated with doxorubicin in the presence or absence of verapamil for 24 hours, followed by the measurement of apoptotic substrates. G) Western blot detection of PARP (both total and cleaved PARP (c-PARP)), total Caspase 3 (Casp3), and cleaved Caspase 3 (c-Casp3) in 231/LSR cells treated with doxorubicin and/or verapamil as above. The relative protein levels of c-PARP, Casp3, and c-Casp3 (signals from both 19 and 17 kD bands) were quantified and are indicated beneath the corresponding blots. (H–J) MDR1 knockdown restores the sensitivity of 231/LSR cells to doxorubicin. Cells were transfected with control siRNA (231/LSR/Non) or MDR1-specific siRNA (231/LSR/siMDR1) for 48 h, followed by treatment with doxorubicin at the indicated concentrations. (H) Protein levels of MDR1 in control and siMDR1-transfected 231/LSR cells. (I) IC 50 values of 231/LSR/Non and 231/LSR/siMDR1 were determined by CCK-8 assay after 4 days of treatment. (J) Western blot analysis of total and cleaved PARP (c-PARP) and Caspase-3 (Casp3, c-Casp3) after 24 h of treatment. Relative protein levels were quantified. Data from (A) and (C) were analyzed using Student’s t-test; IC 50 values from (D) , (E) and (I) were analyzed using Welch’s t-test; data from (F) were analyzed using one-way ANOVA followed by Tukey’s post hoc test. ** p < 0.01.
Article Snippet: After 24 hours of incubation, cells were co-transfected with plasmids encoding the MDR1 promoter-firefly-luciferase (pMDR1–1202, Cat: 37627, Addgene) and the SV40 promoter-Renilla luciferase with luciferase construct (pRL-SV40, Cat: 27163, Addgene) using EndoFectin Transfection Reagents (Cat: EF013, GeneCopoeia).
Techniques: Western Blot, Over Expression, Luciferase, Activity Assay, Transfection, CCK-8 Assay, Software, Inhibition, Enzyme-linked Immunosorbent Assay, Knockdown, Control
Addgene inc is a verified supplier 
