Journal: Scientific Reports

Article Title: The down-regulation of SLC7A11 enhances ROS induced P-gp over-expression and drug resistance in MCF-7 breast cancer cells

doi: 10.1038/s41598-017-03881-9

Figure Lengend Snippet: The differential impact of ADR on GSH/GSSG ratio, the influx of cystine and the expression of SLC7A11 in MCF-7S and MCF-7R cells. ( A ) The baseline ROS level in MCF-7R cells was remarkably higher than in MCF-7S cells. ( B ) ADR reduced the intracellular GSH/GSSG ratio in MCF-7S cells to a comparable level to that of MCF-7R cells, which was reversed by NAC (10 mM, 30 min of pretreatment). ( C ) The utilization of cystine was much higher in the culture medium of MCF-7S cells than that of MCF-7R cells, although both groups were treated with the same initial concentration (1.0 mM). ( D ) ADR inhibited the influx of cystine and the efflux of glutamic acid (substrates of SLC7A11) in MCF-7S (1 μM) and MCF-7R (5 μM) cells. ( E ) The SLC7A11 mRNA level in MCF-7R cells was intrinsically lower than in MCF-7S cells. Moreover, ADR markedly inhibited SLC7A11 mRNA expression in MCF-7S cells (1 μM), but showed a marginal effect on MCF-7R cells (5 μM). ( F ) ADR down-regulated SLC7A11 protein expression in MCF-7S cells. ( G ) The inhibitiory effect of ADR on SLC7A11 mRNA expression showed time-dependence and concentration-dependence in MCF-7R cells. All of the experiments were conducted in triplicate, and data with error bars are presented as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01 vs. control.

Article Snippet: Cells were plated in six-well plates and transfected with siRNAs: SLC7A11 siRNA (h) (sc-76933, Santa Cruz, USA) and Control siRNA-A (sc-37007, Santa Cruz, USA) using the Lipofectamine® RNAiMAX Transfection Reagent (13778150, Life Technologies, USA) and the GibcoTM Opti-MEMTM I Reduced Serum Media (31985-070, Life Technologies, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Concentration Assay, Control

Journal: Scientific Reports

Article Title: The down-regulation of SLC7A11 enhances ROS induced P-gp over-expression and drug resistance in MCF-7 breast cancer cells

doi: 10.1038/s41598-017-03881-9

Figure Lengend Snippet: Down-regulatory effects of SLC7A11 on P-gp expression in MCF-7S cells. ( A ) SLC7A11 mRNA and protein levels were efficiently reduced by siRNA. ( B ) SLC7A11-siRNA increased P-gp mRNA expression, in a negatively dependent way. ( C ) SLC7A11 silence stimulated P-gp expression, shown by immunofluorescence analysis, with flow cytometry. ( D ) SLC7A11 silence increased the intracellular ROS level and P-gp mRNA expression, which could be reversed by NAC (10 mM, 24 h). ( E ) SLC7A11 silence enhanced P-gp protein expression, which could be reversed by NAC (10 mM, 24 h), as shown with laser confocal scanning analysis. Scale bar: 20 µm. ( F ) SLC7A11 silencing enhanced the function of P-gp, which could be reversed by NAC (10 mM, 24 h), and Rho 123 was identified as a typical substrate of P-gp. Scale bar: 100 µm. ( G ) Treatment with an SLC7A11 inhibitor, sulfasalazine, increased ROS levels and P-gp mRNA expression. All of the experiments were conducted in triplicate, and data with error bars are presented as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01 vs. control.

Article Snippet: Cells were plated in six-well plates and transfected with siRNAs: SLC7A11 siRNA (h) (sc-76933, Santa Cruz, USA) and Control siRNA-A (sc-37007, Santa Cruz, USA) using the Lipofectamine® RNAiMAX Transfection Reagent (13778150, Life Technologies, USA) and the GibcoTM Opti-MEMTM I Reduced Serum Media (31985-070, Life Technologies, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: The down-regulation of SLC7A11 enhances ROS induced P-gp over-expression and drug resistance in MCF-7 breast cancer cells

doi: 10.1038/s41598-017-03881-9

Figure Lengend Snippet: Effect of SLC7A11 over-expression on P-gp expression in MCF-7S and MCF-7R cells. ( A ) SLC7A11 over-expression reduced the ROS level in MCF-7S and MCF-7R cells. ( B ) SLC7A11 over-expression inhibited P-gp mRNA expression in MCF-7S and MCF-7R cells. Furthermore, MCF-7S cells that over-expressed SLC7A11 were more sensitive to ADR, compared to MCF-7S cells that has been exposed to a control vector. ( C ) SLC7A11 over-expression increased the cytotoxic sensitivity of ADR on MCF-7S cells. ( D ) SLC7A11 over-expression reduced ROS levels and P-gp mRNA expression in MCF-7S cells, which could be reversed by H 2 O 2 . ( E ) SLC7A11 over-expression decreased the function of P-gp, which could be reversed by H 2 O 2 (0.25 mM, 24 h), as indicated by intracellular Rho 123 accumulation in MCF-7S cells. Scale bar: 100 μm. All of the experiments were conducted in triplicate, and data with error bars are presented as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01 vs. control.

Article Snippet: Cells were plated in six-well plates and transfected with siRNAs: SLC7A11 siRNA (h) (sc-76933, Santa Cruz, USA) and Control siRNA-A (sc-37007, Santa Cruz, USA) using the Lipofectamine® RNAiMAX Transfection Reagent (13778150, Life Technologies, USA) and the GibcoTM Opti-MEMTM I Reduced Serum Media (31985-070, Life Technologies, USA) according to the manufacturer’s instructions.

Techniques: Over Expression, Expressing, Control, Plasmid Preparation

Journal: Scientific Reports

Article Title: The down-regulation of SLC7A11 enhances ROS induced P-gp over-expression and drug resistance in MCF-7 breast cancer cells

doi: 10.1038/s41598-017-03881-9

Figure Lengend Snippet: Multiple factors of ROS generation, modulation of SLC7A11 and supplementary cystine involved in the expression and function of P-gp in MCF-7S cells. ( A ) SLC7A11 silencing greatly promoted P-gp mRNA expression in the presence of H 2 O 2 (0.5 mM, 12 h). ( B ) SLC7A11 silence greatly promoted the protein expression of P-gp in the presence of H 2 O 2 (0.5 mM, 12 h), as shown by flow cytometry analysis. ( C ) SLC7A11 silencing significantly increased the function of P-gp in the presence of H 2 O 2 (0.5 mM, 12 h), as indicated by decreased intracellular Rho 123 accumulation. ( D ) SLC7A11 silencing reduced the cytotoxic effect of H 2 O 2 (24 h) on MCF-7S cells. ( E ) Cystine deprivation (72 h) significantly enhanced the function of P-gp in the presence of H 2 O 2 (0.5 mM, 12 h), as indicated by decreased intracellular Rho 123 accumulation. Scale bar: 100 μm. ( F ) Cystine deprivation (72 h) significantly reduced the cytotoxic effect of H 2 O 2 (24 h) on MCF-7S cells. All of the experiments were conducted in triplicate, and data with error bars are presented as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01 vs. control.

Article Snippet: Cells were plated in six-well plates and transfected with siRNAs: SLC7A11 siRNA (h) (sc-76933, Santa Cruz, USA) and Control siRNA-A (sc-37007, Santa Cruz, USA) using the Lipofectamine® RNAiMAX Transfection Reagent (13778150, Life Technologies, USA) and the GibcoTM Opti-MEMTM I Reduced Serum Media (31985-070, Life Technologies, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: The down-regulation of SLC7A11 enhances ROS induced P-gp over-expression and drug resistance in MCF-7 breast cancer cells

doi: 10.1038/s41598-017-03881-9

Figure Lengend Snippet: A schematic diagram of the reprogrammed homeostasis of GSH synthesis and subsequent P-gp expression of MCF-7S into MCF-7R cells, where ROS, SLC7A11 and cystine play an important role in the process. Red indicates up-regulation and green indicates down-regulation.

Article Snippet: Cells were plated in six-well plates and transfected with siRNAs: SLC7A11 siRNA (h) (sc-76933, Santa Cruz, USA) and Control siRNA-A (sc-37007, Santa Cruz, USA) using the Lipofectamine® RNAiMAX Transfection Reagent (13778150, Life Technologies, USA) and the GibcoTM Opti-MEMTM I Reduced Serum Media (31985-070, Life Technologies, USA) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells

doi: 10.1038/s41598-018-30205-2

Figure Lengend Snippet: CRISPR/Cas9 targeting of KRAS c.35 G > T in SW620 and SW480 cells. ( a ) Indel frequencies in target KRAS region of SW620 and SW480 cells with sgKRAS-G12V, and in HEK293T cells with sgKRAS-WT. ( b ) Major indel patterns detected in the KRAS target region of SW620 cells with sgKRAS-G12V. Blue: sgRNA target sequence; bold blue: c.35 G > T single-nucleotide missense substitution; orange: PAM sequence; red: mismatched nucleotides. ( c ) Cell proliferation assays of indicated cell lines following transduction with lentiviral vector encoding Cas9, Cas9 and sgKRAS-G12V, or Cas9 and sgKRAS-WT. Red: sgRNA sample relevant to tested cell line. Bars represent the mean ± S.E.M. **** (P < 0.0001), ns: not significant.

Article Snippet: One day after seeding, the cells were transfected in GibcoTM Opti-MEMTM I Reduced Serum Medium (Life Technologies, USA) using 1 μg of target lentiviral vector (empty lentiCRISPR v2 or lentiCRISPR v2 containing sgKRAS-WT, sgKRAS-G12V, or sgKRAS-G12D), 0.25 μg of pMD2.G (Addgene, USA), 0.75 μg of psPAX2 (Addgene, USA) and 7 μl of LipofectamineTM 3000 (Life Technologies, USA).

Techniques: CRISPR, Sequencing, Transduction, Plasmid Preparation

Journal: Scientific Reports

Article Title: Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells

doi: 10.1038/s41598-018-30205-2

Figure Lengend Snippet: Indel ratios and transduced cell viability before and after puromycin selection. Indel frequencies in target KRAS region of AsPC-1 and SNU407 cells with sgKRAS-G12D were determined before ( a ) and after ( c ) antibiotic selection. Transduced cell viability was determined using the MTT assay before ( b ) and after ( d ) puromycin selection. For cell proliferation assays, AsPC-1 and SNU407 cells were transfected with lentiviral vectors encoding Cas9, Cas9 and sgKRAS-G12D, or Cas9 and sgKRAS-WT. Red: sgRNA sample relevant to tested cell line. Bars represent the mean ± S.E.M. **** (P < 0.0001), ns: not significant.

Article Snippet: One day after seeding, the cells were transfected in GibcoTM Opti-MEMTM I Reduced Serum Medium (Life Technologies, USA) using 1 μg of target lentiviral vector (empty lentiCRISPR v2 or lentiCRISPR v2 containing sgKRAS-WT, sgKRAS-G12V, or sgKRAS-G12D), 0.25 μg of pMD2.G (Addgene, USA), 0.75 μg of psPAX2 (Addgene, USA) and 7 μl of LipofectamineTM 3000 (Life Technologies, USA).

Techniques: Selection, MTT Assay, Transfection