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. 2023 Oct 13;22(11):e14000. doi: 10.1111/acel.14000

FIGURE 2.

FIGURE 2

Carnitine acetyltransferase (CRAT) knockdown induces cellular senescence and regulates senescence‐associated secretory phenotype (SASP). (a) Cumulative population doublings were determined in human dermal fibroblasts (HDFs) cultured for 3, 6, and 9 days after transfection with siRNA targeting either negative control siRNA (siNC) or CRAT (siCRAT). (b) HDFs transfected with siNC and siCRAT were subjected to senescence‐associated‐β‐galactosidase (SA‐β‐gal) staining 10 days after transfection. Percentage of SA‐β‐gal‐positive cells was quantified (n = 3) and representative photos are shown. Scale bar = 100 μm. (c) The mRNA levels of SASP genes were determined by quantitative real‐time PCR (RT‐PCR) after siNC and siCRAT transfection for 48 h followed by 72 h incubation after media change (n = 5). (d) mRNA expression levels of SASPs of normal fibroblasts cultured for 5 days in conditioned media (CM) from CRAT‐knockdown cells collected 5 days after transfection (n = 3). (e) HDFs were cultured for 10 days in the conditioned media (CM) from CRAT‐knockdown cells, mixed with 10% DMEM in a 1:1 ratio, and then subjected to SA‐β‐gal staining (n = 3). Scale bar = 100 μm. Data are shown as mean ± standard error of mean (SEM). ***p < 0.001, **p < 0.01, *p < 0.05, versus siNC analyzed by t test.