Il 16

Cells were lysed (30 min, 4 °C) with NP40 buffer (10 mM Hepes pH 7.5, 15 mM KCl, 1 mM EGTA, 1 mM EDTA, 1% NP40 and 10% Glycerol) or RIPA buffer (20 mM Tris-HCl pH 7.5, 300 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate and 10% Glycerol), both containing protease and phosphatase inhibitors (20 μM leupeptine, 1.5 μM aprotinin, 1 mM PMSF, 1 mM Na3VO4, 40 mM β-glycerophosphate and 2 mM NaF; all from Sigma-Aldrich). Clarified lysates were quantified with pierce 660 nm protein assay (Thermo Scientific). An equivalent protein amount per sample was analyzed by SDS-PAGE, and proteins were transferred to nitrocellulose membranes (Bio-Rad). Ab used for immunoblot were: rabbit anti -GFP, -ZO-2, -Lin-7A, and -SIPA1L1 (all from Invitrogen) and -IL-16, -syntenin, -MPP1, -GSMD, and -Delphilin (all from Abcam), -GAPDH (Santa-Cruz); mouse anti-tubulin mAb (Sigma), -HA mAb (Covance), and -Scrib (Santa Cruz). Primary Ab recognition was visualized using secondary Ab coupled to fluorescent dyes: anti-rabbit IgG StarBright 700 and anti-mouse IgG StarBright 520 (both from Bio-Rad), and anti-goat IgG IRDye 680 (Li-COR). Blots were analyzed with a ChemiDoc MP Imaging System (Bio-Rad). Densitometric analysis of proteins was performed using ImageJ.

PS-MPs (1 μm) and fluorescent-labelled PS-MPs (1 μm) were purchasedfrom Tianjin Bestra Chromatography Technology Development Center (25 mg/mL stock solution). The stock solution was diluted to the specified concentration using complete medium. The monoclonal antibodies, p16 (ab51243; 1:50 dilution for IHC, 1:1000 dilution for WB), Ki67 (ab15580; 1:200 dilution), γ-H2AX (ab81299; 1:200 dilution), 53BP1 (ab175933; 1:200 dilution), IL-16 (ab180792; 1:100 dilution), 4-Hydroxynonenal (ab48506; 1:100 dilution), H3K9me3 (ab8898; 1:1000 dilution), Histone (ab1791; 1:1000 dilution), IL-6 (ab9324; 1:1000 dilution), IL-8 (ab18672; 1:1000 dilution), TNF-α (ab1793; 1:1000 dilution), were obtained from Abcam (US). The monoclonal antibodies, α-SMA (19,245; 1:500 dilution), p53 (48,818; 1:100 dilution for IHC, 1:1000 dilution for WB), p21 (2947; 1:50 dilution for IHC, 1:1000 dilution for WB), NF-kB (p65) (8242; 1:500 dilution for IHC, 1:1000 dilution for WB), IL-1β (12,242; 1:500 dilution for IF, 1:1000 dilution for WB), β-actin (4970; 1:1000 dilution), were purchased from Cell Signaling Technology (US). Other reagents were purchased from Thermo Fisher, unless otherwise specified.

The cytoplasm and nuclei in LV tissue and RAW264.7 macrophages were isolated using a nuclear separation kit (Njjcbio) according to a previously described protocol [19 (link)]. Briefly, homogenate was collected from LV tissue and cells that were lysed in lysis buffer and further centrifuged at 1000 × g for 10 minutes. The pellets in the bottoms of the tubes contained the nuclei, and the supernatant contained the cytoplasm.
After the cytoplasm, nuclei, LV tissue, and HL-1 cardiomyocytes were, respectively, lysed in RIPA lysis buffer containing 10% protease inhibitors and 10% phosphatase inhibitors, the total protein was obtained and quantitated using a BCA Protein Assay Kit. After separation by electrophoresis on 10% SDS polyacrylamide gels, the proteins were transferred to PVDF membranes. Then, the protein expression of IL-16 (Abcam), Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH (all three from GeneTex) in total LV tissue, the protein expression of p-p65 and GAPDH (both from Abcam) in the cytoplasm, the protein expression of p-p65 and PCNA (GeneTex) in the nuclei, and the protein expression of Bax, Bcl-2, cleaved caspase-3, caspase-3, and GAPDH in HL-1 cardiomyocytes were measured using primary antibodies as indicated in parentheses. After further incubation with the secondary antibodies, the target proteins were detected and analyzed.