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RNAi knockdown of <t>Cdkn1a</t> during senescence in NIT-1 cells. ( A , B ) Viability (gated R3) and transfection efficiency (gated R5) histograms and quantifications of NIT-1 cells transfected with 25 nM siGLO-FITC or mock transfected, 24 h post-transfection. ( C ) qRT-PCR analysis of Cdkn1a or Ppia as a control gene in control or etoposide induced senescent NIT-1 cells (72 h post-etoposide treatment) at 24 h post-transfection with control non-targeting siRNA (siCtrl), or Cdkn1a siRNA. N = 3 or 4 biological replicates per KD, error bars are S.D. ns = not significant, ** p < 0.005, one-way ANOVAs. ( D ) Western blot analysis of p21 on whole cell protein extracts prepared from NIT-1 cells transfected with the indicated siRNAs at 24 h or 72 h post-transfection. Beta-actin was as a loading control. ( E ) Western blot analysis of p21 levels after transfection with control or Cdkn1a siRNA at 72 h post-transfection in control/non-senescent NIT-1 cells in n = 3 biological replicates per transfection. Vinculin was a loading control. Plot shows quantification of p21 normalized to vinculin, error bars are S.D. * p < 0.05 two-tailed t -test. ( F ) Upper plot: viability assay using trypan blue staining and automated cell counting (Bio-Rad TC-20) of control or senescent NIT-1 cells transfected with the indicated siRNAs at 24 h post-transfection. Data are n = 10–12 biological replicates (upper plot), error bars are S.D. Lower plot : viability assay as in upper plot except 48 h following a second transfection which was conducted 2 days after the first transfection in control or senescent NIT-1 cells. Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, *** p < 0.0005, two-tailed T-tests with multiple comparisons corrections ( C , E ) or two-way ANOVA in ( F ).
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RNAi knockdown of <t>Cdkn1a</t> during senescence in NIT-1 cells. ( A , B ) Viability (gated R3) and transfection efficiency (gated R5) histograms and quantifications of NIT-1 cells transfected with 25 nM siGLO-FITC or mock transfected, 24 h post-transfection. ( C ) qRT-PCR analysis of Cdkn1a or Ppia as a control gene in control or etoposide induced senescent NIT-1 cells (72 h post-etoposide treatment) at 24 h post-transfection with control non-targeting siRNA (siCtrl), or Cdkn1a siRNA. N = 3 or 4 biological replicates per KD, error bars are S.D. ns = not significant, ** p < 0.005, one-way ANOVAs. ( D ) Western blot analysis of p21 on whole cell protein extracts prepared from NIT-1 cells transfected with the indicated siRNAs at 24 h or 72 h post-transfection. Beta-actin was as a loading control. ( E ) Western blot analysis of p21 levels after transfection with control or Cdkn1a siRNA at 72 h post-transfection in control/non-senescent NIT-1 cells in n = 3 biological replicates per transfection. Vinculin was a loading control. Plot shows quantification of p21 normalized to vinculin, error bars are S.D. * p < 0.05 two-tailed t -test. ( F ) Upper plot: viability assay using trypan blue staining and automated cell counting (Bio-Rad TC-20) of control or senescent NIT-1 cells transfected with the indicated siRNAs at 24 h post-transfection. Data are n = 10–12 biological replicates (upper plot), error bars are S.D. Lower plot : viability assay as in upper plot except 48 h following a second transfection which was conducted 2 days after the first transfection in control or senescent NIT-1 cells. Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, *** p < 0.0005, two-tailed T-tests with multiple comparisons corrections ( C , E ) or two-way ANOVA in ( F ).
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RNAi knockdown of <t>Cdkn1a</t> during senescence in NIT-1 cells. ( A , B ) Viability (gated R3) and transfection efficiency (gated R5) histograms and quantifications of NIT-1 cells transfected with 25 nM siGLO-FITC or mock transfected, 24 h post-transfection. ( C ) qRT-PCR analysis of Cdkn1a or Ppia as a control gene in control or etoposide induced senescent NIT-1 cells (72 h post-etoposide treatment) at 24 h post-transfection with control non-targeting siRNA (siCtrl), or Cdkn1a siRNA. N = 3 or 4 biological replicates per KD, error bars are S.D. ns = not significant, ** p < 0.005, one-way ANOVAs. ( D ) Western blot analysis of p21 on whole cell protein extracts prepared from NIT-1 cells transfected with the indicated siRNAs at 24 h or 72 h post-transfection. Beta-actin was as a loading control. ( E ) Western blot analysis of p21 levels after transfection with control or Cdkn1a siRNA at 72 h post-transfection in control/non-senescent NIT-1 cells in n = 3 biological replicates per transfection. Vinculin was a loading control. Plot shows quantification of p21 normalized to vinculin, error bars are S.D. * p < 0.05 two-tailed t -test. ( F ) Upper plot: viability assay using trypan blue staining and automated cell counting (Bio-Rad TC-20) of control or senescent NIT-1 cells transfected with the indicated siRNAs at 24 h post-transfection. Data are n = 10–12 biological replicates (upper plot), error bars are S.D. Lower plot : viability assay as in upper plot except 48 h following a second transfection which was conducted 2 days after the first transfection in control or senescent NIT-1 cells. Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, *** p < 0.0005, two-tailed T-tests with multiple comparisons corrections ( C , E ) or two-way ANOVA in ( F ).
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RNAi knockdown of <t>Cdkn1a</t> during senescence in NIT-1 cells. ( A , B ) Viability (gated R3) and transfection efficiency (gated R5) histograms and quantifications of NIT-1 cells transfected with 25 nM siGLO-FITC or mock transfected, 24 h post-transfection. ( C ) qRT-PCR analysis of Cdkn1a or Ppia as a control gene in control or etoposide induced senescent NIT-1 cells (72 h post-etoposide treatment) at 24 h post-transfection with control non-targeting siRNA (siCtrl), or Cdkn1a siRNA. N = 3 or 4 biological replicates per KD, error bars are S.D. ns = not significant, ** p < 0.005, one-way ANOVAs. ( D ) Western blot analysis of p21 on whole cell protein extracts prepared from NIT-1 cells transfected with the indicated siRNAs at 24 h or 72 h post-transfection. Beta-actin was as a loading control. ( E ) Western blot analysis of p21 levels after transfection with control or Cdkn1a siRNA at 72 h post-transfection in control/non-senescent NIT-1 cells in n = 3 biological replicates per transfection. Vinculin was a loading control. Plot shows quantification of p21 normalized to vinculin, error bars are S.D. * p < 0.05 two-tailed t -test. ( F ) Upper plot: viability assay using trypan blue staining and automated cell counting (Bio-Rad TC-20) of control or senescent NIT-1 cells transfected with the indicated siRNAs at 24 h post-transfection. Data are n = 10–12 biological replicates (upper plot), error bars are S.D. Lower plot : viability assay as in upper plot except 48 h following a second transfection which was conducted 2 days after the first transfection in control or senescent NIT-1 cells. Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, *** p < 0.0005, two-tailed T-tests with multiple comparisons corrections ( C , E ) or two-way ANOVA in ( F ).
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RNAi knockdown of <t>Cdkn1a</t> during senescence in NIT-1 cells. ( A , B ) Viability (gated R3) and transfection efficiency (gated R5) histograms and quantifications of NIT-1 cells transfected with 25 nM siGLO-FITC or mock transfected, 24 h post-transfection. ( C ) qRT-PCR analysis of Cdkn1a or Ppia as a control gene in control or etoposide induced senescent NIT-1 cells (72 h post-etoposide treatment) at 24 h post-transfection with control non-targeting siRNA (siCtrl), or Cdkn1a siRNA. N = 3 or 4 biological replicates per KD, error bars are S.D. ns = not significant, ** p < 0.005, one-way ANOVAs. ( D ) Western blot analysis of p21 on whole cell protein extracts prepared from NIT-1 cells transfected with the indicated siRNAs at 24 h or 72 h post-transfection. Beta-actin was as a loading control. ( E ) Western blot analysis of p21 levels after transfection with control or Cdkn1a siRNA at 72 h post-transfection in control/non-senescent NIT-1 cells in n = 3 biological replicates per transfection. Vinculin was a loading control. Plot shows quantification of p21 normalized to vinculin, error bars are S.D. * p < 0.05 two-tailed t -test. ( F ) Upper plot: viability assay using trypan blue staining and automated cell counting (Bio-Rad TC-20) of control or senescent NIT-1 cells transfected with the indicated siRNAs at 24 h post-transfection. Data are n = 10–12 biological replicates (upper plot), error bars are S.D. Lower plot : viability assay as in upper plot except 48 h following a second transfection which was conducted 2 days after the first transfection in control or senescent NIT-1 cells. Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, *** p < 0.0005, two-tailed T-tests with multiple comparisons corrections ( C , E ) or two-way ANOVA in ( F ).
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RNAi knockdown of Cdkn1a during senescence in NIT-1 cells. ( A , B ) Viability (gated R3) and transfection efficiency (gated R5) histograms and quantifications of NIT-1 cells transfected with 25 nM siGLO-FITC or mock transfected, 24 h post-transfection. ( C ) qRT-PCR analysis of Cdkn1a or Ppia as a control gene in control or etoposide induced senescent NIT-1 cells (72 h post-etoposide treatment) at 24 h post-transfection with control non-targeting siRNA (siCtrl), or Cdkn1a siRNA. N = 3 or 4 biological replicates per KD, error bars are S.D. ns = not significant, ** p < 0.005, one-way ANOVAs. ( D ) Western blot analysis of p21 on whole cell protein extracts prepared from NIT-1 cells transfected with the indicated siRNAs at 24 h or 72 h post-transfection. Beta-actin was as a loading control. ( E ) Western blot analysis of p21 levels after transfection with control or Cdkn1a siRNA at 72 h post-transfection in control/non-senescent NIT-1 cells in n = 3 biological replicates per transfection. Vinculin was a loading control. Plot shows quantification of p21 normalized to vinculin, error bars are S.D. * p < 0.05 two-tailed t -test. ( F ) Upper plot: viability assay using trypan blue staining and automated cell counting (Bio-Rad TC-20) of control or senescent NIT-1 cells transfected with the indicated siRNAs at 24 h post-transfection. Data are n = 10–12 biological replicates (upper plot), error bars are S.D. Lower plot : viability assay as in upper plot except 48 h following a second transfection which was conducted 2 days after the first transfection in control or senescent NIT-1 cells. Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, *** p < 0.0005, two-tailed T-tests with multiple comparisons corrections ( C , E ) or two-way ANOVA in ( F ).

Journal: Epigenomes

Article Title: Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21

doi: 10.3390/epigenomes8010010

Figure Lengend Snippet: RNAi knockdown of Cdkn1a during senescence in NIT-1 cells. ( A , B ) Viability (gated R3) and transfection efficiency (gated R5) histograms and quantifications of NIT-1 cells transfected with 25 nM siGLO-FITC or mock transfected, 24 h post-transfection. ( C ) qRT-PCR analysis of Cdkn1a or Ppia as a control gene in control or etoposide induced senescent NIT-1 cells (72 h post-etoposide treatment) at 24 h post-transfection with control non-targeting siRNA (siCtrl), or Cdkn1a siRNA. N = 3 or 4 biological replicates per KD, error bars are S.D. ns = not significant, ** p < 0.005, one-way ANOVAs. ( D ) Western blot analysis of p21 on whole cell protein extracts prepared from NIT-1 cells transfected with the indicated siRNAs at 24 h or 72 h post-transfection. Beta-actin was as a loading control. ( E ) Western blot analysis of p21 levels after transfection with control or Cdkn1a siRNA at 72 h post-transfection in control/non-senescent NIT-1 cells in n = 3 biological replicates per transfection. Vinculin was a loading control. Plot shows quantification of p21 normalized to vinculin, error bars are S.D. * p < 0.05 two-tailed t -test. ( F ) Upper plot: viability assay using trypan blue staining and automated cell counting (Bio-Rad TC-20) of control or senescent NIT-1 cells transfected with the indicated siRNAs at 24 h post-transfection. Data are n = 10–12 biological replicates (upper plot), error bars are S.D. Lower plot : viability assay as in upper plot except 48 h following a second transfection which was conducted 2 days after the first transfection in control or senescent NIT-1 cells. Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, *** p < 0.0005, two-tailed T-tests with multiple comparisons corrections ( C , E ) or two-way ANOVA in ( F ).

Article Snippet: We initially attempted to use a CRISPR-Cas9-based approach for generating constitutive population-level knockout (KO) of Ino80 or Cdkn1a in NIT-1 cells using a commercially available kit (Synthego).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Control, Western Blot, Two Tailed Test, Viability Assay, Staining, Cell Counting

SASP gene activation is unaffected by KD of Cdkn1a in NIT-1 cells Control or senescent NIT-1 cells were transfected with indicated siRNAs and harvested 24 h post-transfection for qRT-PCR analysis of SASP genes Igfbp3 and Serpine1 (encoding Pai1). Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, **** p < 0.0001, two-way ANOVA.

Journal: Epigenomes

Article Title: Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21

doi: 10.3390/epigenomes8010010

Figure Lengend Snippet: SASP gene activation is unaffected by KD of Cdkn1a in NIT-1 cells Control or senescent NIT-1 cells were transfected with indicated siRNAs and harvested 24 h post-transfection for qRT-PCR analysis of SASP genes Igfbp3 and Serpine1 (encoding Pai1). Data are n = 3 or 4 biological replicates, error bars are S.D. ns = not significant, ** p < 0.005, **** p < 0.0001, two-way ANOVA.

Article Snippet: We initially attempted to use a CRISPR-Cas9-based approach for generating constitutive population-level knockout (KO) of Ino80 or Cdkn1a in NIT-1 cells using a commercially available kit (Synthego).

Techniques: Activation Assay, Control, Transfection, Quantitative RT-PCR