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. 2021 Nov 11;298(1):101406. doi: 10.1016/j.jbc.2021.101406

Figure 2.

Figure 2

PELP1 is an inflammatory responsive gene.A, top, FACS analysis of F4/80 (macrophage-specific marker) staining to confirm the macrophage population in the peritoneal fluid; Western blot (middle) and quantitative RT (qRT)–PCR (bottom) analysis of PELP1 in peritoneal macrophages isolated from LPS (10 mg/kg body weight) injected female BALB/c mice (6–8 weeks old) at different time intervals. B, Western blot (top) and qRT–PCR (bottom) analysis of PELP1 in mouse macrophage cells (MMa-bm) treated with LPS (1 μg/ml for 4 h). C, protein (upper) and mRNA (bottom) expression of PELP1 in a human macrophage cell line, MD-CRL9850 treated with LPS (1 μg/ml for 4 h). D, PELP1 mRNA expression level in total zebrafish lysate after intraperitoneal injections with LPS for 12 h at a concentration of 20 μg/fish. E, FACS analysis showing live and necrotic NIH3T3 cells (top) and live and necrotic HC-11 cells (bottom) by 7-AAD staining. Necrosis was induced by repeated freeze–thawing cycles (US stands for unstained). F, Western blot image of PELP1 from RAW 264.7 cells incubated with necrotic NIH3T3 lysate for two different periods; here, LPS served as a positive control for PELP1 induction, and β-actin was utilized as an internal loading control. G, qRT–PCR data of PELP1 mRNA expression levels along with positive control genes. H and I, TNF-α and IL-1β from RAW 264.7 cells incubated with necrotic NIH3T3 lysate for two different periods. J, Western blot image of PELP1 from RAW 264.7 cells incubated with necrotic HC11 lysate for two different periods and LPS as a positive control for PELP1 induction. Vinculin served as an internal loading control. K, qRT–PCR analysis of PELP1 in peritoneal macrophages from necrotic HC11-injected female BALB/c mice of 6 to 8 weeks old. L, Western blot image showing PELP1 expression in RAW 264.7 cells when pretreated with three different NSAIDs, that is, licofelone (10 μM/ml), zileuton (1 μM/ml), and MK886 (5 μg/ml) for 1 h, prior to LPS (1 μg/ml for 4 h) treatment. Actin was used as a control. For all the experiments, the data are represented as mean ± SEM and ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), and ∗∗∗∗ (p < 0.0001) for presenting p values (unless otherwise specified). 7-AAD, 7-aminoactinomycin D; FACS, fluorescent-activated cell sorting; IL-1β, interleukin 1β; LPS, lipopolysaccharide; NSAID, nonsteroidal anti-inflammatory drug; PELP1, proline, glutamic acid, and leucine-rich protein 1; TNF-α, tumor necrosis factor alpha.