Fig 2: rOPN administration elevated the expression of autophagy-related proteins while suppressing apoptosis in rat brain at 24 h after SAH. The effects of rOPN on expression levels of (A) Beclin 1, mean ± SD is 1.016 ± 0.2262 in Sham group, 2.874 ± 1.147 in SAH + Vehicle group, 4.963 ± 2.05 in SAH + rOPN group, F = 15.52, (B) ATG5, mean ± SD is 0.8908 ± 0.2545 in Sham group, 2.332 ± 0.6431 in SAH + Vehicle group, 4.364 ± 1.309 in SAH + rOPN group, F = 25.02, (C) LC3, mean ± SD is 1 ± 0.1845 in Sham group, 1.755 ± 0.3017 in SAH + Vehicle group, 2.7 ± 0.7957 in SAH + rOPN group, F = 17.23, (D) Cleaved Caspase-3, mean ± SD is 1.008 ± 0.186 in Sham group, 4.112 ± 1.528 in SAH + Vehicle group, 2.291 ± 0.6268 in SAH + rOPN group, F = 15.86, (E) Bax, mean ± SD is 1.006 ± 0.321 in Sham group, 37.47 ± 10.86 in SAH + Vehicle group, 23.83 ± 8.143 in SAH + rOPN group, F = 33.13, (F) Bcl-2, mean ± SD is 1.005 ± 0.2736 in Sham group, 0.2309 ± 0.1257 in SAH + Vehicle group, 0.7843 ± 0.2278 in SAH + rOPN group, F = 20.1, in the left hemisphere of rat brain at 24 h after SAH. Sample size is 18, n = 6 per group. Data were presented as mean ± SD. *P < .05, ***P < .001 vs Sham group; #P < .05, ##P < .01 vs SAH + Vehicle group

Fig 3: ER stress activation abrogates protection of FABP4 inhibition against hypoxia-induced apoptosis in cardiomyocytes. (A-C) H9c2 cardiomyocytes were cultured under hypoxia condition in the presence or absence of 50 µM BMS309403 and 10 µM tunicamycin as indicated for 24 h. (A) The protein expressions of GPR78 and CHOP were determined by Western blot analysis. ß-actin is the internal control. The quantification analysis of protein expression is shown in the right. Student's t test (n = 3). **, P < 0.01. NS, not significant. (B) The cell apoptosis was assessed by the flow cytometric analysis combined with annexin V-FITC and PI double-staining assay. The annexin V-positive cells are defined as apoptotic cells, and the percentage of apoptotic cells is depicted in the right. Student's t test (n = 4). **, P < 0.01. NS, not significant. (C) The protein expressions of Bax, Bcl-2 and cleaved caspase-3 were determined by Western blot analysis. ß-actin is the internal control. The quantification analysis of protein expression is shown in the right. Student's t test (n = 3). **, P < 0.01. NS, not significant

Fig 4: ANRIL protects against H/R injury. (A-E) H9c2 cells were transiently transfected with pcDNA3.1-vector or pcDNA3.1-ANRIL plasmids (2 or 4 µg per well). At 2 days after transfection, cells were cultured under normoxia or exposed to H/R (4 h hypoxia followed by 8 h reoxygenation). (A) ANRIL expression was determined via reverse transcription-quantitative PCR analysis. ß-actin served as an internal control. Results are expressed as relative to normoxia group. (B) The release of LDH from cells was measured and expressed as a percentage of the total LDH activity (n=5). (C and D) Apoptosis was detected using a TUNEL assay and defined as a percentage of TUNEL positive cells (green) of the total number of cells (DAPI, blue) (n=12). Scale bar, 100 µm. (E) The expression levels of Bax, Bcl-2 and cleaved caspase-3 were determined using an immunoblotting assay (n=3). Data are presented as the mean ± SD. **P<0.01. NS, not significant; LDH, lactate dehydrogenase; ANRIL, antisense non-coding RNA in the INK4 locus; H/R, hypoxia/reoxygenation.

Fig 5: FABP4 inhibitor BMS309403 protects against hypoxia-induced apoptosis in cardiomyocytes. (A) H9c2 cardiomyocytes were cultured under normoxia or hypoxia condition for 24 h in the presence or absence of 50 µM BMS309403. The cell apoptosis was evaluated by the flow cytometric analysis combined with annexin V-FITC and PI double-staining assay. The annexin V-positive cells are defined as apoptotic cells, and the percentage of apoptotic cells is depicted in the right. Student's t test (n = 4). **, P < 0.01. (B) H9c2 cardiomyocytes were treated as in (A). The protein expressions of Bax, Bcl-2 and cleaved caspase-3 were determined by Western blot analysis. ß-actin is the internal control. The quantification analysis of protein expression is shown in the right. Student's t test (n = 3). **, P < 0.01