Journal: Aging Cell

Article Title: Clearance of senescent cells during cardiac ischemia–reperfusion injury improves recovery

doi: 10.1111/acel.13249

Figure Lengend Snippet: Cardiac ischemia–reperfusion induces cellular senescence. (a) Experimental design. Mice were either subjected to 60 minutes LAD‐ligation followed by reperfusion (IR) or received no injury (Control) and hearts collected at the indicated times. (b) At 4 weeks post‐IR, hearts were collected and the LV region apical to suture isolated. Real‐time qPCR gene expression analysis performed to determine the relative expression of p16 and p21 mRNA (normalized to GADPH). N ≥ 4/group. (c) SA‐β‐Gal staining in control and at 24 hours, 72 hours, and 1 week post‐IR. Lower panels) Higher magnification images of the peri‐infarct region. Scale bars 50 µm. (d) Representative images of TAF, γH2AX co‐localized with Telomere immuno‐FISH, in a trop‐C + CM (trop‐C white, telo‐FISH red, γH2AX green). White arrows indicate individual TAF. Images are obtained from the z‐stacks of 10 μm sections. Right image panel) Higher magnification of 5 regions showing 6 individual TAF. The merged image demonstrates the co‐localization of γH2AX (green) and telomere (red). Individual red and green channels are also shown. Scale bar 2.5 μm. Right) The mean number of CMs with ≥5‐TAF at 1 and 4 weeks post‐IR. N = 3/group. (e) Left) Representative image of p16 expression at 4 weeks post‐IRI (p16 red, trop‐C green, DAPI blue. ( i ) shows a magnified example of a p16 + CM (trop‐C + ), and ( ii ) is an example of a p16 + interstitial cell. Scale bar 20 µm. Right) Quantification of the percentage p16 expressing cells at 4 weeks post‐IR N ≥ 3/group. Data are mean ± SEM. B and E were analyzed by 2‐tailed unpaired t‐test; D was analyzed by a 1‐way ANOVA followed by Tukey's post hoc test, ** p < 0.01; * p < 0.05

Article Snippet: Primary antibodies used: rat ant‐p21 (HUGO291, Abcam ab107099), goat‐anti‐troponin C (Abcam, ab30807), rabbit anti‐p16 (Rockland, 100‐401‐170) and rat anti‐CD31 (MEC13.3, BD Biosciences, 550274).

Techniques: Ligation, Isolation, Expressing, Staining

Journal: Aging Cell

Article Title: Clearance of senescent cells during cardiac ischemia–reperfusion injury improves recovery

doi: 10.1111/acel.13249

Figure Lengend Snippet: Pharmacological clearance of senescent cells following ischemia–reperfusion reduces cellular senescence and improves cardiac function. (a) Mice were either subjected to 60‐minute IR or received no injury (Control). After IR mice were randomly assigned to the vehicle (IR) or navitoclax (IR+Nav) groups and provided with navitoclax or vehicle daily from 4 days post‐IR for 7 days. MRI was performed at 5 weeks post‐IR, and hearts were collected. (b) Representative images of trop‐C + CMs co‐expressing p16 (examples indicated by white arrows) and p16 expressing intestinal cells (examples indicated by yellow arrows) in the LV peri‐infarct region (p16 red, trop‐C green and DAPI blue). Scale bars 20 µm. (c) Quantification of the percentage trop‐C + CMs and intestinal cells expressing p16 + in the LV peri‐infarct region. N ≥ 3/group. (d) Representative images of trop‐C + CMs co‐expressing p21 in the LV peri‐infarct region (p21 red, trop‐C green, and DAPI blue). White arrows indicate examples of p16 + CMs. Scale bars 20 µm. (e) Quantification of the percentage of trop‐C + CMs co‐expressing p21 + in the LV peri‐infarct region. N = 3/group. (f) Examples of individual short‐axis cine‐MR images of mouse hearts. Scale bars 5 mm. G and (h) EF%, EDV and ESV were calculated based on manual measurements of LV epicardial and endocardial borders. Measurements were made in all cine slices at end‐diastole and end‐systole. Graphs representing data obtained from MRI analysis. Control N = 6, IR N = 12 and IR+Nav N = 13. Data are mean±SEM. c, e, g, and H were analyzed by one‐way ANOVA followed by Tukey's post hoc test, ** p < 0.01; * p < 0.05

Article Snippet: Primary antibodies used: rat ant‐p21 (HUGO291, Abcam ab107099), goat‐anti‐troponin C (Abcam, ab30807), rabbit anti‐p16 (Rockland, 100‐401‐170) and rat anti‐CD31 (MEC13.3, BD Biosciences, 550274).

Techniques: Expressing

Journal:

Article Title: Inactivation of both the Retinoblastoma Tumor Suppressor and p21 by the Human Papillomavirus Type 16 E7 Oncoprotein Is Necessary To Inhibit Cell Cycle Arrest in Human Epithelial Cells

doi: 10.1128/JVI.76.20.10559-10568.2002

Figure Lengend Snippet: Induction of HMEC proliferation by E7. (A) HMEC passage number relative to time in culture. Times of transduction and G418 selection are indicated. The first passage after drug selection (passage 4) was designated week 0. (B) Micrographs showing HMEC morphologies at different time points. The passage number is indicated on each image. (C) Western blots probed with anti-p16 (Pharmingen), anti-p21 (Ab-2; Calbiochem), anti-cyclin A (BF-683; Pharmingen), anti-cdc25a (F-6; Santa Cruz Biotechnology), and antinucleolin (as a loading control, C23; Santa Cruz Biotechnology). (D) Western blot probed with an anti-Rb antibody (G3-245; Pharmingen) comparing Rb levels in E7-expressing HMECs with that of untransduced, pre-M0 HMECs (first lane).

Article Snippet: The passage number is indicated on each image. (C) Western blots probed with anti-p16 (Pharmingen), anti-p21 (Ab-2; Calbiochem), anti-cyclin A (BF-683; Pharmingen), anti-cdc25a (F-6; Santa Cruz Biotechnology), and antinucleolin (as a loading control, C23; Santa Cruz Biotechnology). (D) Western blot probed with an anti-Rb antibody (G3-245; Pharmingen) comparing Rb levels in E7-expressing HMECs with that of untransduced, pre-M0 HMECs (first lane).

Techniques: Transduction, Selection, Western Blot, Expressing