Journal: Aging Cell

Article Title: Age‐Dependent KLK8 Upregulation Contributes to Elevated Susceptibility to Ventilator‐Induced Lung Injury in the Elderly Mice

doi: 10.1111/acel.70304

Figure Lengend Snippet: KLK8 activates PARP1/2 via inactivation of the fibronectin/FAK/Akt signaling pathway, thus promoting pulmonary endothelial senescence. (A) Flowchart of CMAP platform analysis. Top 150 genes positively and negatively correlated with age in human and mouse lungs, and differentially expressed genes (DEGs) in senescent endothelial cells were queried in the Connectivity Map (CMap) platform to identify candidate drugs. (B) MLVECs were seeded on plates coated with 5 μg/cm 2 fibronectin. Twenty‐four hours later, MLVECs were treated with Ad‐KLK8 (MOI 10) or Ad‐vector (MOI 10) for 72 h. Western blot showing PARylation levels and p‐Akt/Akt protein levels in MLVECs ( n = 3). (C) MLVECs were seeded on plates coated with 5 μg/cm 2 fibronectin. Twenty‐four hours later, MLVECs were treated with Ad‐KLK8 (MOI 10) with or without the FAK inhibitor (FAKi) Defactinib (5 μM) for 72 h. Western blot showing PARylation levels and p‐Akt/Akt protein levels in MLVECs ( n = 3). (D, E) MLVECs were treated with vehicle or olaparib (1, 5, 10 μM). Twenty‐four hours later, MLVECs were treated with Ad‐KLK8 at an MOI of 10 for 72 h ( n = 3). (D) Western blot showing p53 and p21 protein levels in MLVECs. E, Representative SA‐β‐gal staining. Scale bar, 100 μm. (F, G) Young mice (2 months) were instilled intratracheally with Ad‐KLK8 or Ad‐vector. Seventy hours later, lung tissues were harvested for analysis ( n = 6). (F) Western blot showing p‐FAK/FAK and p‐Akt/Akt protein levels in lung tissues. (G) Western blot showing PARylation levels in lung tissues. (H) Western blot showing p‐FAK/FAK and p‐Akt/Akt protein levels in lung tissues of young (13–40 years old) and aged (66–76 years old) humans ( n = 6). (I) Western blot showing PARylation levels of young and aged humans ( n = 6). (J) Western blot showing p‐FAK/FAK and p‐Akt/Akt protein levels in lung tissues of young (2 months) and aged (18 months) KLK8 +/+ and KLK8 −/− mice ( n = 6). (K) Western blot showing PARylation levels of young and aged KLK8 +/+ and KLK8 −/− mice ( n = 6). (L) Schematic diagram of the mechanism underlying KLK8‐induced pulmonary endothelial cell senescence. KLK8 cleaves extracellular matrix (ECM) protein fibronectin, leading to the inhibition of the fibronectin‐FAK‐Akt signaling pathway and subsequent PARP1/2 activation, contributing to pulmonary endothelial cell senescence. Data were presented as means ± SEM. “You” represents “Young.”

Article Snippet: The PARP1/2 inhibitor olaparib (HY‐10162, MCE, Shanghai, China) was used at a concentration of 1, 5, and 10 μM to inhibit MLVEC senescence.

Techniques: Plasmid Preparation, Western Blot, Staining, Inhibition, Activation Assay

Journal: Aging Cell

Article Title: Age‐Dependent KLK8 Upregulation Contributes to Elevated Susceptibility to Ventilator‐Induced Lung Injury in the Elderly Mice

doi: 10.1111/acel.70304

Figure Lengend Snippet: The PARP1/2 inhibitor alleviates pulmonary endothelial senescence in both intra‐pulmonary KLK8‐overexpressing mice and aged mice. (A–C) Young mice (2 months) were intraperitoneally injected with vehicle or the PARP1/2 inhibitor (PARPi) olaparib (5 mg/kg) in saline containing 0.1% DMSO every other day. Fourteen days later, they were intratracheally transfected with Ad‐KLK8 (1 × 10 8 pfu). Seventy‐two hours after Ad‐KLK8 transfection, lung tissues were harvested for analysis. (A) Timeline of the experimental procedures. (B) Western blot showing p53 and p21 protein levels in lung tissues. Corresponding histograms were shown on the right of representative protein bands. (C) Representative senescent markers staining showing p53 (red) expression in lung sections. Endothelial cells were immunostained with anti‐CD31 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. The percentage of CD31 + p53 + cell numbers in total CD31 + cells was shown on the right. (D–F) Aged mice were intraperitoneally injected with vehicle or olaparib (5 mg/kg) in saline containing 0.1% DMSO every other day. Thirty days later, lung tissues were harvested for western blot analysis and immunofluorescence. (D) Timeline of the experimental procedures. (E) Western blot showing p53 and p21 protein levels in lung tissues. Corresponding histograms were shown on the right of representative protein bands. (F) Representative senescent markers staining showing p53 (red) expression in lung sections. Endothelial cells were immunostained with anti‐CD31 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. The percentage of CD31 + p53 + cell numbers in total CD31 + cells was shown on the right. Data were presented as means ± SEM ( n = 6). ** p < 0.01.

Article Snippet: The PARP1/2 inhibitor olaparib (HY‐10162, MCE, Shanghai, China) was used at a concentration of 1, 5, and 10 μM to inhibit MLVEC senescence.

Techniques: Injection, Saline, Transfection, Western Blot, Staining, Expressing, Immunofluorescence

Journal: Aging Cell

Article Title: Age‐Dependent KLK8 Upregulation Contributes to Elevated Susceptibility to Ventilator‐Induced Lung Injury in the Elderly Mice

doi: 10.1111/acel.70304

Figure Lengend Snippet: The PARP1/2 inhibitor mitigates the elevated susceptibility to low‐magnitude mechanical stretch in KLK8‐overexpressing MLVECs, both intra‐pulmonary KLK8‐overexpressing mice and aged mice. (A–C) MLVECs were treated with vehicle or olaparib (1, 5, 10 μM). Twenty‐four hours later, MLVECs were treated with Ad‐KLK8 at an MOI of 10. Seventy‐two hours later, MLVECs were subjected to physiological, low‐magnitude (7%) cyclic stretch (CS) for 4 h ( n = 3). (A) Western blot showing VE‐cadherin and VCAM‐1 protein levels in MLVECs. Corresponding histograms were shown on the right of representative protein bands. (B), MLVECs were seeded on Collagen I coated Bioflex culture plates and the FITC fluorescence was detected as described in Materials and Methods. FITC fluorescence signal was visualized by fluorescence microscopy. (C) Quantification of FITC fluorescence signal by using ImageJ. Original magnification, × 200. Scale bar = 200 μm. (D–G) Young mice were intraperitoneally injected with vehicle or the PARP1/2 inhibitor olaparib (5 mg/kg) in saline containing 0.1% DMSO every other day. Fourteen days later, they were intratracheally transfected with Ad‐KLK8 (1 × 10 8 pfu). Seventy‐two hours later, mice with intra‐pulmonary KLK8 overexpression that were treated with either vehicle or olaparib were subjected to low‐tidal‐volume (LTV) mechanical ventilation (8 mL/kg) for 4 h. Lung tissues were harvested for analysis ( n = 6). (D) Timeline of the experimental procedures. (E) Western blot showing VE‐cadherin and VCAM‐1 protein levels in mice. (F) The left lower lung was used for histological evaluation by H&E staining. Original magnification, × 200. Scale bar = 100 μm. (G) The severity of lung injury was scored by two pathologists blinded to group allocation. (H–K) Aged mice were intraperitoneally injected with vehicle or olaparib (5 mg/kg) in saline containing 0.1% DMSO every other day. Thirty days later, aged mice treated with either vehicle or olaparib were subjected to LTV mechanical ventilation (8 mL/kg) for 4 h ( n = 6). (H) Timeline of the experimental procedures. (I) Western blot showing VE‐cadherin and VCAM‐1 protein levels in lung tissues. (J) The left lower lung was used for histological evaluation by H&E staining. Original magnification, × 200. Scale bar = 100 μm. (K) The severity of lung injury was scored by two pathologists blinded to group allocation. Data were presented as means ± SEM. ** p < 0.01.

Article Snippet: The PARP1/2 inhibitor olaparib (HY‐10162, MCE, Shanghai, China) was used at a concentration of 1, 5, and 10 μM to inhibit MLVEC senescence.

Techniques: Western Blot, Fluorescence, Microscopy, Injection, Saline, Transfection, Over Expression, Staining

Journal: bioRxiv

Article Title: ATM and 53BP1 regulate alternative end joining-mediated V(D)J recombination

doi: 10.1101/2024.04.25.591195

Figure Lengend Snippet: ( A ) Coding and signal prey junctions of Vκ region DSBs from the Jκ1CE bait are aggregated in a resection window of ±100 bps around the RAG1/2 DSB (RSS). Representative plots showing restricted (DNA2i treatment), extended (Polα inhibition; XR1 deletion; Parp1 and ATM inhibition or deletion), or no significant change (53BP1 deletion) are indicated. ( B-D ) The percentage of junctions that enriched in the window of ±10 bps around RSS site was used as indicator for resection, as cyan dashed rectangle in (A). The significance of enrichment changes when combined with indicated inhibitors or gene modification or both were evaluated by one-way ANOVA plus post comparison (B-C) and two-way ANOVA plus post comparison (D); N=3.

Article Snippet: The following compounds and their intended effects are indicated: G1/G0 arrest – STI-571 (3μM, TCI Chemicals, Cat # TCI0936-100MG); Cell cycle analysis – EdU (50μM, Cayman Cat # 20518); Inducible gene expression – Doxycycline (2μM, Sigma-Aldrich, Cat # D9891-10G); Parp inhibition – Olaparib (Parpi #1, 10μM ( , ), AdooQ BioScience, Cat # A10111) and Talazoparib (Parpi #2, 1μM ( , ), ApexBio Cat # A4153); Polθ inhibition – Novobiocin (Polθi #1, 100μM , BioVision, Cat # B1526-1G) and ART558 (Polθi #2, 10μM , MedChem Express, Cat # HY-141520); Polα inhibition – Adarotene (Polαi #1, 1μM ( , ), MedChem Express, Cat # HY-14808) and CD437 (Polαi #2, 5μM , Sigma-Aldrich, Cat # 178496-5MG); ATR inhibition – AZD6738 (ATRi, 0.5μM , MedChem Express, Cat # HY-19323); ATM inhibition – Ku60019 (ATMi, 2μM , Sigma-Aldrich, Cat # SML1416-5MG); DNA2 inhibition – C5 (DNA2i, 20μM , MedChem Express, Cat # HY-128729); Mre11 inhibition – PFM01 (Mre11eni #1, 10μM), PFM03 (Mre11eni #2, 10μM) and PFM39 (Mre11exi, 100μM) were from the Tainer lab ( ).

Techniques: Inhibition, Modification, Comparison