Journal: Scientific Reports

Article Title: Serum deprivation initiates adaptation and survival to oxidative stress in prostate cancer cells

doi: 10.1038/s41598-020-68668-x

Figure Lengend Snippet: ( a ) Serum deprivation protected cancer cells from apoptosis during oxidative stress. (i) One million (1 × 10 6 ) PC3 prostate cancer cells were harvested with or without serum, then were treated with 250 μM H 2 O 2 , and/or 10 mM n-acetyl-cysteine (NAC) or 2 μM Doxorubicin (Doxo). Apoptosis was measured via annexin V-FITC/PI double staining and flow cytometry. Q1 (Annexin V+ , PI+): dead cells; Q2 (Annexin V-, PI+): late stage apoptosis cells; Q3 (Annexin V+, PI−): early stage apoptosis cells; and Q4 (Annexin V− , PI−): live cells. (ii) A graphical representation of live cells. (iii) A graphical representation of total apoptotic cells (a sum of dead, early- and late-stages). Images were acquired via flow cytometry (Accuri C6 Cytometer; BD Biosciences); data was analyzed using FlowJo (v10). The mean ± SEM of data were obtained from three independent replicate experiments. Statistical was analysis (one-way ANOVA) was done with GraphPad Prism (*** p < 0.001; ** p < 0.01; * p < 0.05). ( b ) Serum deprivation protected cancer cells from apoptosis during oxidative stress. (i) One million (1 × 10 6 ) DU145 prostate cancer cells were harvested with or without serum, then were treated with 250 μM H 2 O 2 , and/or 10 mM n-acetyl-cysteine (NAC) or 2 μM Doxorubicin (Doxo). Apoptosis was measured via annexin V-FITC/PI double staining and flow cytometry. Q1 (Annexin V+ , PI+): dead cells; Q2 (Annexin V−, PI+): late stage apoptosis cells; Q3 (Annexin V+ , PI−): early stage apoptosis cells; and Q4 (Annexin V− , PI−): live cells. (ii) A graphical representation of live cells. (iii) A graphical representation of total apoptotic cells (a sum of dead, early- and late-stages). Images were acquired via flow cytometry (Accuri C6 Cytometer; BD Biosciences); data was analyzed using FlowJo (v10). The mean ± SEM of data were obtained from three independent replicate experiments. Statistical analysis ( one-way ANOVA ) was done with GraphPad Prism (*** p < 0.001; ** p < 0.01; * p < 0.05). ( c ) Serum deprivation protected cancer cells from apoptosis during oxidative stress. (i) One million (1 × 10 6 ) LNCaP prostate cancer cells were harvested with or without serum, then were treated with 250 μM H 2 O 2 , and/or 10 mM n-acetyl-cysteine (NAC) or 2 μM Doxorubicin (Doxo). Apoptosis was measured via annexin V-FITC/PI double staining and flow cytometry. Q1 (Annexin V+ , PI+): dead cells; Q2 (Annexin V−, PI+): late stage apoptosis cells; Q3 (Annexin V+ , PI −): early stage apoptosis cells; and Q4 (Annexin V− , PI−): live cells. (ii) A graphical representation of live cells. (iii) A graphical representation of total apoptotic cells (a sum of dead, early- and late-stages). Images were acquired via flow cytometry (Accuri C6 Cytometer; BD Biosciences); data was analyzed using FlowJo (v10). The mean ± SEM of data were obtained from three independent replicate experiments. Statistical analysis ( one-way ANOVA ) was done with GraphPad Prism (*** p < 0.001; ** p < 0.01; * p < 0.05).

Article Snippet: Annexin-V Apoptosis Detection Kit (Cell Signaling Technology) was used to quantify the levels of apoptosis according to the manufacturer's instructions.

Techniques: Double Staining, Flow Cytometry, Cytometry

Journal: Scientific Reports

Article Title: Serum deprivation initiates adaptation and survival to oxidative stress in prostate cancer cells

doi: 10.1038/s41598-020-68668-x

Figure Lengend Snippet: RelA/p65 (NF-κB) knockdown promotes apoptosis in response to oxidative stress. Two hundred thousand (2 × 10 ) DU145 prostate cancer cells were plated in a 6-well dish and transfected with 100 nM human siRNA targeting RelA/p65 or nonspecific control siRNA for 24 h, followed by recovery in 10% FBS/RPMI for an additional 24 h. Cells were harvested for western blotting and apoptosis. ( a , b ) RelA/p65 was utilized to demonstrate that siRNA was specific as well as β-actin was used as a loading control to demonstrate equal amounts of protein was used for this assay. ( c ) Control siRNA efficiency was measured via fluorescence. Pictures of gels/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure . ( d , e ) Cells were harvested with or without serum, then were treated with 250 μM H 2 O 2 , and/or 10 mM n-acetyl-cysteine (NAC) or 2 μM Doxorubicin (Doxo). One hundred thousand cells (1 × 10 ) cells were measured via annexin V-FITC/PI double staining and flow cytometry. Q1 (Annexin V+ , PI+): dead cells; Q2 (Annexin V−, PI+): late stage apoptosis cells; Q3 (Annexin V+ , PI −): early stage apoptosis cells; and Q4 (Annexin V− , PI−): live cells. ( f ) A graphical representation of live cells. ( g ) A graphical representation of total apoptotic cells. Images were acquired via flow cytometry (Accuri C6 Cytometer; BD Biosciences); data was analyzed using FlowJo (v10). The mean ± SEM of data were obtained from three independent replicate experiments. Statistical analysis ( one-way ANOVA ) was done with GraphPad Prism (*** p < 0.001; ** p < 0.01; * p < 0.05).

Article Snippet: Annexin-V Apoptosis Detection Kit (Cell Signaling Technology) was used to quantify the levels of apoptosis according to the manufacturer's instructions.

Techniques: Transfection, Western Blot, Fluorescence, Expressing, Double Staining, Flow Cytometry, Cytometry

Journal: Cancer Science

Article Title: Interleukin‐6 production mediated by the IRE 1‐ XBP 1 pathway confers radioresistance in human papillomavirus‐negative oropharyngeal carcinoma

doi: 10.1111/cas.14094

Figure Lengend Snippet: Schematic model of IRE 1‐ XBP 1‐induced radioresistance via interleukin‐6 ( IL ‐6) upregulation. Exposure to X‐ray irradiation causes endoplasmic reticulum stress (ERS) , and increased levels of IRE 1 promote the splicing of XBP 1 mRNA . The activated transcription factor XBP 1s translocates to the nucleus and induces the expression of IL ‐6. Concurrently, the formation of DNA double‐strand breaks (DSB) is inhibited and helps decrease apoptosis, which is related to radioresistance

Article Snippet: Experiments were performed according to the protocol supplied with the Annexin‐Green Apoptosis cell detection reagent kit (Cell Signaling Technology).

Techniques: Irradiation, Expressing

Journal: Scientific Reports

Article Title: Multipronged activity of combinatorial miR-143 and miR-506 inhibits Lung Cancer cell cycle progression and angiogenesis in vitro

doi: 10.1038/s41598-018-28872-2

Figure Lengend Snippet: The combinatorial miR treatment induced strong cytotoxic and apoptotic effect, along with Caspase activation ( A ) Potential synergism is illustrated by the combination’s (Combo) cytotoxicity being larger than the addition of the cytotoxicity of the individual miRs alone. Fluorescence intensity, which correlates to number of dead cells, was measured till 33 h post transfection. Lipofectamine with Scramble (Lipo), and untreated cells (Control) were used as negative controls. Positive control (Positive) was provided by the vendor. *p < 0.05 of combo vs. Control, miR-143, miR-506. ( B ) FACS Annexin V/PI analysis of cell apoptosis treated with individual mimics, and combination at 24 and 48 h compared to untreated cells. ( C ) Caspase-3/7 activity of A549 cells treated with individual and combination of miR, at 24 h. Cells without any treatment (Negative control-NC), with Lipofectamine, and with no-target control miRNA (scramble) were used as experimental controls. Doxorubicin (DOX, equimolar) was used as positive control. **p < 0.01 relative to NC. ( D ) Annexin V positive A549 cells, following transfection with miR-143 and/or miR-506 at 24 and 48 h post transfection.

Article Snippet: We evaluated the capacity of the combinatorial treatment to induce apoptotic activity against A549 lung cancer cells, using a Annexin V/PI Early Apoptosis Detection Kit (Cell Signaling), following an established protocol .

Techniques: Activation Assay, Fluorescence, Transfection, Positive Control, Activity Assay, Negative Control

Journal: Scientific Reports

Article Title: Multipronged activity of combinatorial miR-143 and miR-506 inhibits Lung Cancer cell cycle progression and angiogenesis in vitro

doi: 10.1038/s41598-018-28872-2

Figure Lengend Snippet: Annexin V/PI apoptosis analysis of A549 cell populations, as determined by flow-cytometric quantification (Average of triplicates ± SEM).

Article Snippet: We evaluated the capacity of the combinatorial treatment to induce apoptotic activity against A549 lung cancer cells, using a Annexin V/PI Early Apoptosis Detection Kit (Cell Signaling), following an established protocol .

Techniques:

Journal: Scientific Reports

Article Title: Multipronged activity of combinatorial miR-143 and miR-506 inhibits Lung Cancer cell cycle progression and angiogenesis in vitro

doi: 10.1038/s41598-018-28872-2

Figure Lengend Snippet: Genes and their activity on the cell cycle, which demonstrated >10% up-/down-regulation, as identified by the microarray analysis of A549 cells transfected with miR-143/506, for 24 and/or 48 h post transfection, compared to control.

Article Snippet: We evaluated the capacity of the combinatorial treatment to induce apoptotic activity against A549 lung cancer cells, using a Annexin V/PI Early Apoptosis Detection Kit (Cell Signaling), following an established protocol .

Techniques: Activity Assay, Microarray, Transfection, Over Expression, Migration