Journal: Nature communications

Article Title: Cdkn1b overexpression in adult mice alters the balance between genome and tissue aging

doi: 10.1038/ncomms3626

Figure Lengend Snippet: Panel A shows western blots for pp53(ser18) (a full-length image of this blot is shown in Supplementary Figure 10, panel B) and actin in lung, intra-abdominal fat (IA Fat), and skin of R26-M2rtTA transgenic mice that either carry or do not carry the TRE-Tight-Cdkn1b transgene as indicated in the figure where, starting at two months of age, mice were continuously treated with 1 mg/ml doxycycline for two months and assessed at 4 months of age. Additionally, Cdkn1b expression in the same tissues from a control 24 month old mouse is included for comparison. Arrows indicate the positions of size markers in kDa. Panels B and C show immune-fluorescence staining for γ-H2AX in epidermal sections of control (B) and Cdkn1b over-expressing (C) mice. Panels D and E show immune-fluorescence staining for TP53BP1 in epidermal sections of control (D) and Cdkn1b over-expressing (E) mice. Panels F and G demonstrate reduced frequency of γ-H2AX (n = 9, error bars indicate s.d., ρ = 0.04, two-tailed t-test) and TP53BP1 (n = 10, error bars indicate s.d , ρ = 3.7 × 10 −5 , two-tailed t-test) foci, respectively, in epidermal sections from Cdkn1b over-expressing mice relative to controls. Panel H shows SA-β galactosidase staining of intra-abdominal fat (IAT) for R26-M2rtTA control and R26-M2rtTA;TRE-Tight-Cdkn1b transgenic mice that were treated with 1 mg/ml doxycycline either from birth through 3 months of age (left panels) or starting at 2 months of age and through 8 months of age (right panels). Bars = 5 mm. Quantitative assessment of SA-β galactosidase activity was made using ONPG as a substrate and results from this assay are shown below each panel. Additionally, the weight of total IAT recovered from each animal is given in grams (g). Qualitatively similar SA-β galactosidase staining was observed in a two additional sets of control and R26-M2rtTA;TRE-Tight-Cdkn1b mice treated with 0.25 mg/ ml doxycycline from months 2–7 although quantitative measures were not performed.

Article Snippet: Following immunoblotting (western blot) Cdkn1b, pp53(ser18), and actin were detected using anti-p27(C-19) (1/1000, Santa Cruz Biotechnology Cat No sc-528), anti-pp53(ser18) (1/1000, Cell Signaling Cat No 9284S), or anti-actin (1/1000, Sigma Cat No A2066) primary antibodies and HRP conjugated anti-rabbit secondary antibody (1/2000, Mllipore AP132P GtxRb IgG (H+L) HRP).

Techniques: Western Blot, Transgenic Assay, Expressing, Control, Comparison, Fluorescence, Staining, Two Tailed Test, Activity Assay

Journal: Cancers

Article Title: Regulation of PD-L1 Expression by SAHA-Mediated Histone Deacetylase Inhibition in Lung Cancer Cells

doi: 10.3390/cancers17172919

Figure Lengend Snippet: Mechanisms of PD-L1 regulation in lung cancer. PD-L1 expression in tumor cells is regulated at multiple levels. The p53–p21 axis can negatively influence PD-L1 transcription, while oncogenic pathways such as PI3K/AKT and JAK/STAT promote its up-regulation. Epigenetic mechanisms play a critical role: histone acetylation and methylation dynamically regulate chromatin accessibility, and DNA methylation (e.g., via DNMT1) can repress or activate PD-L1 transcription depending on context. Collectively, these mechanisms contribute to PD-L1-mediated immune evasion and tumor survival.

Article Snippet: pp53 (Phospho-p53) , CST , 12571 , 1:1000.

Techniques: Expressing, Methylation, DNA Methylation Assay

Journal: Oncotarget

Article Title: Selective antitumor activity of roscovitine in head and neck cancer.

doi: 10.18632/oncotarget.9560

Figure Lengend Snippet: Figure 2: Roscovitine treatment activates p53 and induces p53-dependent suppression of HPV-positive cells growth. A. HPV+ UMSCC47 cells were transiently transfected with control or p53 shRNA and plated for survival after the treatment with increasing doses of roscovitine; standard deviations are calculated from two independent experiments. B. Relative mRNA levels of p53 target gene CDKN1A (p21) in HPV-positive SCC090 cells treated or not with 20 μM roscovitine for 24 hours; standard deviations are calculated from two independent experiments. C. SCC090 cells were treated or not with roscovitine, lysed, and immunoblotted with indicated antibodies. D. HPV-positive UMSCC47 and SCC090 cells were treated or not with roscovitine; the cells were collected and HPV16 mRNA levels were determined in qRT-PCR (top), or p53 protein levels were determined in Western Blot (bottom). E. HPV-positive SCC090 cells were treated for 24 hours with roscovitine, or zeocin, or left untreated as a control, lysed and immunoblotted with indicated antibodies.

Article Snippet: Membranes were blocked with 3% BSA in PBS and incubated with antibodies against ƳH2AX and pATM (Abcam), p53 and p21 (Santa Cruz), pp53 Ser15 (Cell Signaling) and tubulin (Sigma).

Techniques: Transfection, Control, shRNA, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: Selective antitumor activity of roscovitine in head and neck cancer

doi: 10.18632/oncotarget.9560

Figure Lengend Snippet: A. Survival after increasing doses of roscovitine was determined in HPV-negative SCC35, SCC61, FaDu and UNC-7 (labeled in black) and HPV+ UMSCC47, SCC104 and SCC090 (labeled in grey) head and neck cancer cell lines; standard deviations are calculated from four independent experiments. B. HPV- SCC61 and HPV+ UMSCC47 cells expressing either control, or p53 shRNAs, were treated with 20μM of roscovitine; immunoblotting with indicated antibodies was performed 24 hours after the treatment. C. HPV+ SCC090 cells transfected with control or p53 shRNA were treated with roscovitine for 24h and immunoblotted with ϒH2AX antibody. D. Two HPV- cells lines, FaDu and SCC35, were treated with 20μM of roscovitine for 24 and 48 hours and immunoblotted with indicated antibodies.

Article Snippet: Membranes were blocked with 3% BSA in PBS and incubated with antibodies against ΥH2AX and pATM (Abcam), p53 and p21 (Santa Cruz), pp53 Ser15 (Cell Signaling) and tubulin (Sigma).

Techniques: Labeling, Expressing, Control, Western Blot, Transfection, shRNA

Journal: Oncotarget

Article Title: Selective antitumor activity of roscovitine in head and neck cancer

doi: 10.18632/oncotarget.9560

Figure Lengend Snippet: A. HPV+ UMSCC47 cells were transiently transfected with control or p53 shRNA and plated for survival after the treatment with increasing doses of roscovitine; standard deviations are calculated from two independent experiments. B. Relative mRNA levels of p53 target gene CDKN1A (p21) in HPV-positive SCC090 cells treated or not with 20 μM roscovitine for 24 hours; standard deviations are calculated from two independent experiments. C. SCC090 cells were treated or not with roscovitine, lysed, and immunoblotted with indicated antibodies. D. HPV-positive UMSCC47 and SCC090 cells were treated or not with roscovitine; the cells were collected and HPV16 mRNA levels were determined in qRT-PCR ( top ), or p53 protein levels were determined in Western Blot ( bottom ). E. HPV-positive SCC090 cells were treated for 24 hours with roscovitine, or zeocin, or left untreated as a control, lysed and immunoblotted with indicated antibodies.

Article Snippet: Membranes were blocked with 3% BSA in PBS and incubated with antibodies against ΥH2AX and pATM (Abcam), p53 and p21 (Santa Cruz), pp53 Ser15 (Cell Signaling) and tubulin (Sigma).

Techniques: Transfection, Control, shRNA, Quantitative RT-PCR, Western Blot