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Loss of <t>αSNAP</t> triggers detachment of human intestinal epithelial cells. Control SK-CO15 cells ( SK-neo ) and cells stably expressing <t>siRNA-resistant</t> bovine αSNAP ( SK -α SNAP ) were transfected with either control or two human αSNAP-specific siRNA duplexes ( D1 and D2 ). A, expression of αSNAP and its binding partner NSF was determined 72 h post-transfection. B and C , cell detachment was examined by counting adhered and total number of cells using bright field microscopy 72 h post-transfection. Data are presented as the mean ± S.E. ( n = 3); *, p
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1) Product Images from "N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *"

Article Title: N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.498691

Loss of αSNAP triggers detachment of human intestinal epithelial cells. Control SK-CO15 cells ( SK-neo ) and cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) were transfected with either control or two human αSNAP-specific siRNA duplexes ( D1 and D2 ). A, expression of αSNAP and its binding partner NSF was determined 72 h post-transfection. B and C , cell detachment was examined by counting adhered and total number of cells using bright field microscopy 72 h post-transfection. Data are presented as the mean ± S.E. ( n = 3); *, p
Figure Legend Snippet: Loss of αSNAP triggers detachment of human intestinal epithelial cells. Control SK-CO15 cells ( SK-neo ) and cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) were transfected with either control or two human αSNAP-specific siRNA duplexes ( D1 and D2 ). A, expression of αSNAP and its binding partner NSF was determined 72 h post-transfection. B and C , cell detachment was examined by counting adhered and total number of cells using bright field microscopy 72 h post-transfection. Data are presented as the mean ± S.E. ( n = 3); *, p

Techniques Used: Stable Transfection, Expressing, Transfection, Binding Assay, Microscopy

Effect of αSNAP depletion on ECM adhesion and integrin processing are independent from induction of apoptosis. SK-CO15 cells were transfected with either control or two αSNAP-specific siRNA duplexes. 24 h later these cells were treated for an additional 48 h with either vehicle or a pan-caspase inhibitor, Q-VAD-OPH. Cells were examined for apoptosis induction and β1 integrin maturation ( A ), β1 integrin localization ( B ), and cell attachment to ECM ( C ). Arrows indicate plasma membrane labeling of β1 integrin in control SK-CO15 cells, whereas arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells treated and untreated with pan-caspase inhibitor. Data are presented as the mean ± S.E. ( n = 3); *, p
Figure Legend Snippet: Effect of αSNAP depletion on ECM adhesion and integrin processing are independent from induction of apoptosis. SK-CO15 cells were transfected with either control or two αSNAP-specific siRNA duplexes. 24 h later these cells were treated for an additional 48 h with either vehicle or a pan-caspase inhibitor, Q-VAD-OPH. Cells were examined for apoptosis induction and β1 integrin maturation ( A ), β1 integrin localization ( B ), and cell attachment to ECM ( C ). Arrows indicate plasma membrane labeling of β1 integrin in control SK-CO15 cells, whereas arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells treated and untreated with pan-caspase inhibitor. Data are presented as the mean ± S.E. ( n = 3); *, p

Techniques Used: Transfection, Cell Attachment Assay, Labeling

Inhibitory anti- β 1 integrin antibody, but not siRNA-mediated integrin depletion, inhibits epithelial ECM adhesion and invasion. A and B , SK-CO15 cells were transfected with control, β1 integrin, β4 integrin siRNAs, or co-transfected with siRNAs against these integrins and αSNAP. Expression of adhesion proteins and cell attachment to the substrate were examined 72 h post-transfection. C–E , control ( SK-neo ) and αSNAP-overexpressing ( SK -α SNAP ) epithelial cells were preincubated with either β1 integrin-inhibitory antibody, MAB13, or the isotype-matched control antibody. Cell adhesion to collagen I ( C ) and invasion into Matrigel ( D and E ) was examined as described under “Experimental Procedures.” Data are presented as the mean ± S.E. ( n = 3); *, p
Figure Legend Snippet: Inhibitory anti- β 1 integrin antibody, but not siRNA-mediated integrin depletion, inhibits epithelial ECM adhesion and invasion. A and B , SK-CO15 cells were transfected with control, β1 integrin, β4 integrin siRNAs, or co-transfected with siRNAs against these integrins and αSNAP. Expression of adhesion proteins and cell attachment to the substrate were examined 72 h post-transfection. C–E , control ( SK-neo ) and αSNAP-overexpressing ( SK -α SNAP ) epithelial cells were preincubated with either β1 integrin-inhibitory antibody, MAB13, or the isotype-matched control antibody. Cell adhesion to collagen I ( C ) and invasion into Matrigel ( D and E ) was examined as described under “Experimental Procedures.” Data are presented as the mean ± S.E. ( n = 3); *, p

Techniques Used: Transfection, Expressing, Cell Attachment Assay

Loss of αSNAP disrupts structure of focal adhesions and affects expression and processing of key FA proteins. Control and αSNAP siRNA-transfected SK-CO15 cells were fixed 72 h post-transfection and FA were visualized by immunofluorescence labeling of vinculin and confocal microscopy ( A ). Arrows show intact vinculin-based FA structures in control cells, whereas arrowheads indicate diffuse vinculin labeling in αSNAP-depleted cells. Scale bar , 20 μm. Expression and phosphorylation of major transmembrane, scaffolding, and signaling FA proteins in control and αSNAP-depleted epithelial cells was determined by immunoblotting analysis ( B ) with densitometric quantification ( C ). Data are presented as the mean ± S.E. ( n = 3); *, p
Figure Legend Snippet: Loss of αSNAP disrupts structure of focal adhesions and affects expression and processing of key FA proteins. Control and αSNAP siRNA-transfected SK-CO15 cells were fixed 72 h post-transfection and FA were visualized by immunofluorescence labeling of vinculin and confocal microscopy ( A ). Arrows show intact vinculin-based FA structures in control cells, whereas arrowheads indicate diffuse vinculin labeling in αSNAP-depleted cells. Scale bar , 20 μm. Expression and phosphorylation of major transmembrane, scaffolding, and signaling FA proteins in control and αSNAP-depleted epithelial cells was determined by immunoblotting analysis ( B ) with densitometric quantification ( C ). Data are presented as the mean ± S.E. ( n = 3); *, p

Techniques Used: Expressing, Transfection, Immunofluorescence, Labeling, Confocal Microscopy, Scaffolding

The effects of αSNAP depletion on β 1 integrin and other FA proteins can be rescued by expression of siRNA-resistant αSNAP. SK-CO15 cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) and their empty vector controls ( SK-neo ) were transfected with either control or αSNAP-specific siRNAs. A, localization of β1 integrin was determined by immunofluorescence labeling and confocal microscopy 72 h post-transfection. B, expression and processing of different FA proteins was determined by immunoblotting. Arrows indicate plasma membrane labeling of β1 integrin in control or αSNAP-siRNA-transfected and rescued cells. Arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells without rescue. Scale bar , 20 μm.
Figure Legend Snippet: The effects of αSNAP depletion on β 1 integrin and other FA proteins can be rescued by expression of siRNA-resistant αSNAP. SK-CO15 cells stably expressing siRNA-resistant bovine αSNAP ( SK -α SNAP ) and their empty vector controls ( SK-neo ) were transfected with either control or αSNAP-specific siRNAs. A, localization of β1 integrin was determined by immunofluorescence labeling and confocal microscopy 72 h post-transfection. B, expression and processing of different FA proteins was determined by immunoblotting. Arrows indicate plasma membrane labeling of β1 integrin in control or αSNAP-siRNA-transfected and rescued cells. Arrowheads show intracellular localization of β1 integrin in αSNAP-depleted cells without rescue. Scale bar , 20 μm.

Techniques Used: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Immunofluorescence, Labeling, Confocal Microscopy

2) Product Images from "Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells"

Article Title: Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2018.02.009

BRAF and BCORL1 mutations in resistant cells. (A) Whole-exome sequencing copy number variation analysis revealed focal imbalances of the distal region of chromosome 7 in A375-R1 cells compared to parental cells. BRAF locus (zoomed area) lies within the amplified region but shows loss of exons 2 to 8. (B) Sanger sequencing of the deleted BRAF allele showing in-frame junction of exon 1 with exon 9 in A375-R1 cells. (C) PCR amplification of the region across the break point reveals the presence of two BRAF transcripts in A375-R1 cells: full-length wild-type (1216-bp band) and truncated (213-bp band). (D) Anti-BRAF Western blotting shows the presence of a smaller band of approximately 47kDa in A375-R1 cells. Actin is shown as a loading control. (E) siRNA-mediated silencing of BRAF in A375-R1 cells leads to downregulation of both full-length and truncated BRAF proteins, suppression of MEK1/2 phosphorylation, and decrease in cell viability. (F) Whole-exome sequencing comparative analysis of A375-R1 versus A375 cells revealed acquired mutations in the four indicated genes at frequency > 35%. SIFT prediction of mutation impact on protein function is shown. (G) Sanger validation of BCORL1 heterozygous genomic mutation in A375-R1 cells.
Figure Legend Snippet: BRAF and BCORL1 mutations in resistant cells. (A) Whole-exome sequencing copy number variation analysis revealed focal imbalances of the distal region of chromosome 7 in A375-R1 cells compared to parental cells. BRAF locus (zoomed area) lies within the amplified region but shows loss of exons 2 to 8. (B) Sanger sequencing of the deleted BRAF allele showing in-frame junction of exon 1 with exon 9 in A375-R1 cells. (C) PCR amplification of the region across the break point reveals the presence of two BRAF transcripts in A375-R1 cells: full-length wild-type (1216-bp band) and truncated (213-bp band). (D) Anti-BRAF Western blotting shows the presence of a smaller band of approximately 47kDa in A375-R1 cells. Actin is shown as a loading control. (E) siRNA-mediated silencing of BRAF in A375-R1 cells leads to downregulation of both full-length and truncated BRAF proteins, suppression of MEK1/2 phosphorylation, and decrease in cell viability. (F) Whole-exome sequencing comparative analysis of A375-R1 versus A375 cells revealed acquired mutations in the four indicated genes at frequency > 35%. SIFT prediction of mutation impact on protein function is shown. (G) Sanger validation of BCORL1 heterozygous genomic mutation in A375-R1 cells.

Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Western Blot, Mutagenesis

Stable knockdown of BCORL1 in A375 cells (A-C) and A375-p47BRAF V600E [clone E9] (D-F). (A, D) Efficiency of shRNA-mediated BCORL1 silencing as shown by quantitative PCR using GAPDH as a reference gene. (B, E) Dose-response curves obtained in the presence of increasing concentrations of vemurafenib, with cells expressing a nontargeting ( shNT , blue curves) or a BCORL1-specific ( shBCORL1 , red curves) shRNA. (C, F) CRISPR/Cas9 system was used to disrupt BCORL1 gene; vemurafenib dose-response curves are shown comparing knockout ( KO ) with parental ( WT ) cells. (G) CRISPR/Cas9-mediated gene editing was used to introduce the Q1076H substitution in the endogenous BCORL1 locus; vemurafenib dose-response curves are shown comparing two mutated clones ( C8 and D7 ) with a wild-type clone ( WT ) and with parental A375 cells ( Par ). All curves are representative of at least three experiments. For all panels, extra-sum-of-squares F test was run to compare the two curves; P values are indicated at the lower-left corner, where P
Figure Legend Snippet: Stable knockdown of BCORL1 in A375 cells (A-C) and A375-p47BRAF V600E [clone E9] (D-F). (A, D) Efficiency of shRNA-mediated BCORL1 silencing as shown by quantitative PCR using GAPDH as a reference gene. (B, E) Dose-response curves obtained in the presence of increasing concentrations of vemurafenib, with cells expressing a nontargeting ( shNT , blue curves) or a BCORL1-specific ( shBCORL1 , red curves) shRNA. (C, F) CRISPR/Cas9 system was used to disrupt BCORL1 gene; vemurafenib dose-response curves are shown comparing knockout ( KO ) with parental ( WT ) cells. (G) CRISPR/Cas9-mediated gene editing was used to introduce the Q1076H substitution in the endogenous BCORL1 locus; vemurafenib dose-response curves are shown comparing two mutated clones ( C8 and D7 ) with a wild-type clone ( WT ) and with parental A375 cells ( Par ). All curves are representative of at least three experiments. For all panels, extra-sum-of-squares F test was run to compare the two curves; P values are indicated at the lower-left corner, where P

Techniques Used: shRNA, Real-time Polymerase Chain Reaction, Expressing, CRISPR, Knock-Out, Introduce, Clone Assay

Functional validation of mutations. (A-C) Transient co-transfection of p47BRAF V600E and BCORL1 Q1076H conferred partial resistance to vemurafenib-mediated inhibition of A375 cell growth (A) and MAPK signaling (C). Forty-eight hours after transfection, the cells were challenged for additional 48 (A) or 4 (C) hours with vemurafenib and harvested. Cell proliferation shown in A was detected by thymidine incorporation. (B) Histogram plot from two proliferation experiments (mean ± SEM). MAPK pathway activity shown in C was detected by Western blotting as MEK and ERK phosphorylation. (D-G) Stably transfected A375 clones expressing HA-tagged p47BRAF V600E (D, Western blot, clone E9) and wild-type or mutated BCORL1 (E, qPCR), singularly or combined, were isolated and tested in proliferation assays for vemurafenib sensitivity. Data from at least four independent experiments are reported as mean ± SEM IC50 values (F); the red bar represents A375-R1 cells, for comparison; ev, empty vector. (G) Representative Western blot showing MEK1/2 phosphorylation (pMEK) in transfected cells treated with the indicated vemurafenib doses for 4 hours. Actin is shown as a control. (H) Correlation between p47BRAF V600E expression ( x -axis, log scale) determined by p47-specific qPCR and vemurafenib IC50 ( y -axis, log scale). Clone E9, used for all experiments, is indicated. (I) Upper panel: siRNA-mediated silencing of BCORL1 ( siBCORL1 ) and BRAF ( siBRAF ) in A375-R1 cells; a nontargeting scrambled siRNA ( siNT ) was used as a control. Lysates were probed with the indicated antibodies. Lower panel: Parental A375 cells were transiently transfected with empty vector, wild-type ( WT ), or mutated ( Q1076H ) BCORL1 and checked for BRAF expression. Actin is shown for loading control.
Figure Legend Snippet: Functional validation of mutations. (A-C) Transient co-transfection of p47BRAF V600E and BCORL1 Q1076H conferred partial resistance to vemurafenib-mediated inhibition of A375 cell growth (A) and MAPK signaling (C). Forty-eight hours after transfection, the cells were challenged for additional 48 (A) or 4 (C) hours with vemurafenib and harvested. Cell proliferation shown in A was detected by thymidine incorporation. (B) Histogram plot from two proliferation experiments (mean ± SEM). MAPK pathway activity shown in C was detected by Western blotting as MEK and ERK phosphorylation. (D-G) Stably transfected A375 clones expressing HA-tagged p47BRAF V600E (D, Western blot, clone E9) and wild-type or mutated BCORL1 (E, qPCR), singularly or combined, were isolated and tested in proliferation assays for vemurafenib sensitivity. Data from at least four independent experiments are reported as mean ± SEM IC50 values (F); the red bar represents A375-R1 cells, for comparison; ev, empty vector. (G) Representative Western blot showing MEK1/2 phosphorylation (pMEK) in transfected cells treated with the indicated vemurafenib doses for 4 hours. Actin is shown as a control. (H) Correlation between p47BRAF V600E expression ( x -axis, log scale) determined by p47-specific qPCR and vemurafenib IC50 ( y -axis, log scale). Clone E9, used for all experiments, is indicated. (I) Upper panel: siRNA-mediated silencing of BCORL1 ( siBCORL1 ) and BRAF ( siBRAF ) in A375-R1 cells; a nontargeting scrambled siRNA ( siNT ) was used as a control. Lysates were probed with the indicated antibodies. Lower panel: Parental A375 cells were transiently transfected with empty vector, wild-type ( WT ), or mutated ( Q1076H ) BCORL1 and checked for BRAF expression. Actin is shown for loading control.

Techniques Used: Functional Assay, Cotransfection, Inhibition, Transfection, Activity Assay, Western Blot, Stable Transfection, Clone Assay, Expressing, Real-time Polymerase Chain Reaction, Isolation, Plasmid Preparation

Related Articles

Concentration Assay:

Article Title: Altered MicroRNA Processing in Heritable Pulmonary Arterial Hypertension
Article Snippet: .. Transfections were performed using DharmaFECT-1 with gene-specific siRNA pools (Dharmacon, Lafayette, CO) at a final concentration of 100 nM. siGENOME nontargeting siRNA Pool-2 (Dharmacon) was used as a negative control. ..

Transfection:

Article Title: Altered MicroRNA Processing in Heritable Pulmonary Arterial Hypertension
Article Snippet: .. Transfections were performed using DharmaFECT-1 with gene-specific siRNA pools (Dharmacon, Lafayette, CO) at a final concentration of 100 nM. siGENOME nontargeting siRNA Pool-2 (Dharmacon) was used as a negative control. ..

Article Title: Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells
Article Snippet: .. Gene-specific siRNA pools (siGENOME SMARTpool; BCORL1, #M-019215-01; BRAF, #M-003460-03; RAF1, #M-003601-02) and control nontargeting siRNA (#D-001210-01) were purchased from Dharmacon and used for transient transfections following manufacturer's recommendations. .. For the generation of stably silenced cell lines, a BCORL1-specific mission shRNA plasmid clone targeting the 3′-UTR was purchased from Sigma-Aldrich (#TRCN0000033474).

Negative Control:

Article Title: Altered MicroRNA Processing in Heritable Pulmonary Arterial Hypertension
Article Snippet: .. Transfections were performed using DharmaFECT-1 with gene-specific siRNA pools (Dharmacon, Lafayette, CO) at a final concentration of 100 nM. siGENOME nontargeting siRNA Pool-2 (Dharmacon) was used as a negative control. ..

Expressing:

Article Title: N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells *
Article Snippet: .. Individual siRNA duplexes, GAAGGUGGCUGGUUACGCU (duplex 1) and CAGAGUUGGUGGACAUCGA (duplex 2; Dharmacon, Lafayette, CO), were used to down-regulate αSNAP expression, whereas knockdown of other targets was performed by using gene-specific siRNA pools purchased either from Dharmacon or Santa Cruz Biotechnology (Dallas, TX). .. A noncoding siRNA duplex-2 (Dharmacon) served as a control.

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    Horizon Discovery sirna experiments gene expression
    Cisplatin induces intrinsic and ROS dependent apoptosis. a Division rate was determined by counting cells on 4 consecutive days. b , c Viable (Annexin V negative/PI negative) cells after co-incubation with b 2 mM Glutathion or c 50 µM zVAD-fmk and 10 µM cisplatin for 48 h. ( d , bottom) Western blot analysis of <t>BAX</t> and BAK expression in cells transfected with <t>siRNA</t> targeting BAX and BAK or control siRNA and ( d , top) flow cytometric analysis of viability (Annexin V negative/PI negative) after treatment with 10 µM cisplatin for 48 h. Data represent means ± SD from at least three independent experiments
    Sirna Experiments Gene Expression, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cisplatin induces intrinsic and ROS dependent apoptosis. a Division rate was determined by counting cells on 4 consecutive days. b , c Viable (Annexin V negative/PI negative) cells after co-incubation with b 2 mM Glutathion or c 50 µM zVAD-fmk and 10 µM cisplatin for 48 h. ( d , bottom) Western blot analysis of BAX and BAK expression in cells transfected with siRNA targeting BAX and BAK or control siRNA and ( d , top) flow cytometric analysis of viability (Annexin V negative/PI negative) after treatment with 10 µM cisplatin for 48 h. Data represent means ± SD from at least three independent experiments

    Journal: Cell Death & Disease

    Article Title: Direct impact of cisplatin on mitochondria induces ROS production that dictates cell fate of ovarian cancer cells

    doi: 10.1038/s41419-019-2081-4

    Figure Lengend Snippet: Cisplatin induces intrinsic and ROS dependent apoptosis. a Division rate was determined by counting cells on 4 consecutive days. b , c Viable (Annexin V negative/PI negative) cells after co-incubation with b 2 mM Glutathion or c 50 µM zVAD-fmk and 10 µM cisplatin for 48 h. ( d , bottom) Western blot analysis of BAX and BAK expression in cells transfected with siRNA targeting BAX and BAK or control siRNA and ( d , top) flow cytometric analysis of viability (Annexin V negative/PI negative) after treatment with 10 µM cisplatin for 48 h. Data represent means ± SD from at least three independent experiments

    Article Snippet: siRNA experiments Gene expression of BAX, BAK, PGC1α, TFAM, or UCP2 was silenced using siGENOME SMARTpool siRNA (Horizon Discovery Ltd, Cambridge, GB).

    Techniques: Incubation, Western Blot, Expressing, Transfection, Flow Cytometry

    Reduction of mtROS enhances survival after cisplatin treatment. a – c Flow cytometric analysis of OVCAR-3 and OVCAR-4 after co-treatment with ATP synthase inhibitor Oligomycin A (5 µM) and 10 µM cisplatin for 48 h. a Oligomycin A reduces cisplatin mediated mtROS induction (MitoSOX red) while b mitochondrial content (Mitotracker Green) is unaffected. c Oligomycin A increases viability (Annexin V negative/PI negative) after cisplatin incubation. d Western blot analysis of OVCAR-3 and OVCAR-8 cells after treatment with 10 µM cisplatin for 48 h shows increased TFAM protein expression. e – g siRNA mediated knockdown of PGC1α in OVCAR-4. e Expression of PGC1α (top) and TFAM (bottom) are decreased in transfected OVCAR-4 cells. f Flow cytometric analysis shows decreased mtROS induction (MitoSox red) in PGC1α silenced cells after treatment with 10 µM cisplatin for 48 h. g Flow cytometric analysis of cell viability (Annexin V negative/PI negative) reveals PGCA1 α knockdown mediated enhanced survival after treatment with 10 µM cisplatin for 48 h. ( h , left) siRNA mediated knockdown of TFAM in OVCAR-4 cells ( h , right) is associated with enhanced viability (Annexin V negative/PI negative) in a flow cytometric analysis after treatment with 10 µM cisplatin for 48 h. Data represent means ± SD or a representative experiment from at least three independent experiments

    Journal: Cell Death & Disease

    Article Title: Direct impact of cisplatin on mitochondria induces ROS production that dictates cell fate of ovarian cancer cells

    doi: 10.1038/s41419-019-2081-4

    Figure Lengend Snippet: Reduction of mtROS enhances survival after cisplatin treatment. a – c Flow cytometric analysis of OVCAR-3 and OVCAR-4 after co-treatment with ATP synthase inhibitor Oligomycin A (5 µM) and 10 µM cisplatin for 48 h. a Oligomycin A reduces cisplatin mediated mtROS induction (MitoSOX red) while b mitochondrial content (Mitotracker Green) is unaffected. c Oligomycin A increases viability (Annexin V negative/PI negative) after cisplatin incubation. d Western blot analysis of OVCAR-3 and OVCAR-8 cells after treatment with 10 µM cisplatin for 48 h shows increased TFAM protein expression. e – g siRNA mediated knockdown of PGC1α in OVCAR-4. e Expression of PGC1α (top) and TFAM (bottom) are decreased in transfected OVCAR-4 cells. f Flow cytometric analysis shows decreased mtROS induction (MitoSox red) in PGC1α silenced cells after treatment with 10 µM cisplatin for 48 h. g Flow cytometric analysis of cell viability (Annexin V negative/PI negative) reveals PGCA1 α knockdown mediated enhanced survival after treatment with 10 µM cisplatin for 48 h. ( h , left) siRNA mediated knockdown of TFAM in OVCAR-4 cells ( h , right) is associated with enhanced viability (Annexin V negative/PI negative) in a flow cytometric analysis after treatment with 10 µM cisplatin for 48 h. Data represent means ± SD or a representative experiment from at least three independent experiments

    Article Snippet: siRNA experiments Gene expression of BAX, BAK, PGC1α, TFAM, or UCP2 was silenced using siGENOME SMARTpool siRNA (Horizon Discovery Ltd, Cambridge, GB).

    Techniques: Flow Cytometry, Incubation, Western Blot, Expressing, Transfection