Prohibitin 1 regulates mtDNA release and downstream inflammatory responses (2022)
Figure 1

Figure 1. Loss of Phb1 induces inflammation in vivo and in vitro

(A) After randomized, 8-12 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (30 mg/kg) for 12 hours via intraperitoneal injection. Serum was collected and ELISA assays were used to detect the level of IL-1β (n = 6, 10, 6, and 12 mice for each group, respectively). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p< 0.0001 (Student's t-test).

(B) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated by 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed and the culture medium was collected. Western blotting (WB) assays were used to detect protein levels in whole cell lysates (Lys) and culture medium (Sup).

(C) BMDMs were extracted from Phb1F/F or Phb1MyeKO mice and stimulated with 20% culture supernatant of L929 cells for 7 days. Differentiated BMDMs were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. ELISA assays were used to detect protein levels in the culture medium. Data of BMDMs from the separate mice (n = 6 mice for each group) and are presented as mean ± SEM. *** p < 0.001 (Student's t-test).

(D) After randomized, 4 weeks old and sex-matched Phb1F/F mice and Phb1MyeKO mice were administered with PBS or LPS (300 mg/kg) to induce septic shock via intraperitoneal injection. The life span of Phb1F/F mice (n = 6) and Phb1MyeKO mice (n = 7) was recorded.

(E) PBMCs were extracted from normal controls and patients with sepsis (n = 6 individuals). Intracellular mRNA levels were detected by real-time quantitative PCR (RT-qPCR) assays (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01 (Student's t-test).

(F) PBMCs were extracted from normal controls and infant patients with sepsis. Protein levels in whole cell lysates were detected by WB assays.

(G-H) J774A.1 cells were stimulated with LPS (200 ng/ml) for 6 hours or recombinant IL-1β (100 pg/ml) for 24 hours. Intracellular mRNA levels were detected by RT-qPCR assays. Data are from four independent experiments for (G) and six independent experiments for (H) (mean ± SEM). n.s., no significance, ** p < 0.01, *** p < 0.001, **** p< 0.0001 (Student's t-test).

(I) After J774A.1 cells were stimulated with LPS (200 ng/ml) for 0, 3, 6, 12 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.

(J) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the cells were lysed, and the culture medium was collected. Protein levels in whole cell lysates (Lys) and culture medium (Sup) were detected by WB assays.

(K) After J774A.1 cells with Phb1 or Phb2 knockdown were stimulated with LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes, the culture medium was collected. ELISA assays were used to detect IL-1β levels in the culture medium. Data are from three independent experiments (mean ± SEM). n.s., no significance, **** p < 0.0001 (Student's t-test).