Fig 2: PHLDA3 inhibition alleviates ERS-induced apoptosis during H/R injury. (A) Cell viability was assessed using Cell Counting Kit-8 assays following H/R injury. The release of (B) LDH and (C) CK following H/R was detected by ELISA. (D) Apoptotic cells were measured by flow cytometry. (E) Expression of ERS-associated proteins, including GRP78, CHOP and cleaved caspase-12 was detected using western blotting. The left panel shows representative blots and the right panel shows the quantitative results. Data are presented as the mean ± standard deviation. n=4/group. *P<0.05 vs. AdshRNA or AdshPHLDA3 + control. #P<0.05 vs. AdshRNA + H/R group. PHLDA3, pleckstrin homology-like domain family A member 3; ERS, endoplasmic reticulum stress; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; GRP78, 78 kDa glucose-regulated protein; AdshRNA, adenovirus encoding scrambled short hairpin RNA; AdshPHLDA3, adenoviral vectors encoding PHLDA3 shRNA; 7-AAD, 7-aminoactinomycin D; Cle cas-12, cleaved caspase-12; CK, creatine kinase.

Fig 3: Analysis of exosomal proteins from isolated exosomes by UC, TEI, DTS chip, PLL-MNPs, PEI-MNPs, and LF-bis-MPA-MNPs (LF-MNPs). (a) Expression of exosomal protein markers (CD63, CD9, and CD81), proteins indicative of the interaction between exosomes and LF (GAPDH, GAG), cancer-associated protein marker (Hsp60), microvesicle protein marker (ARF6), and apoptotic bodies protein marker (Grp78) in EVs isolated by UC, TEI, DTS chip, PLL-MNPs, PEI-MNPs, and LF-bis-MPA-MNPs (LF-MNPs). (b) Purity of EV determined by the ratio of EV particle concentration (NTA) to protein concentration measured by the Bradford assay. Each bar represents the mean ± standard deviation (n = 3, * p-value = 0.05)

Fig 4: CVB3 stimulation could not directly elicit ER stress in macrophages.(A) GRP78 expression in RAW264.7 cells after stimulated with various doses of CVB3 for 24 h. (B) GRP78 expression in RAW264.7 cells after stimulated with CVB3 at a dose of 10 MOI for various hours. (C) GRP78 expression in BMDMs after stimulated with CVB3 at a dose of 10 MOI for various hours. (D) GRP78 expression in splenic macrophages after stimulated with CVB3 at a dose of 10 MOI in vitro for 24 h. RAW264.7 cells were infected with CVB3, inactivated CVB3 or a recombinant infectious CVB3 expressing GFP (CVB3-GFP) respectively at a dose of MOI = 10 for 24 h, and following removal of the inoculum, cells were collected and the level of CVB3 structural protein VP1 was detected by immunofluorescence assays (E), and the amounts of positive- (F) and negative-(G) strand RNAs of CVB3 were detected by real-time PCR. RAW264.7 cells, BMDMs or myocardiocytes were infected with CVB3 at 10 p.f.u. per cell and following removal of the inoculum, amounts of progeny virus were determined over 24 h (H). Individual experiment was conducted 3 times with similar results. **P < 0.01, ***P < 0.001, n.s., no significance.

Fig 5: TIM induced apoptosis through the NOXA/MCL-1 axis and triggering ER stress. U2OS cells were treated with 4 nM TIM for 48 h. (A) TIM treatment decreased the protein expression of MCL-1, but increased the protein expression of NOXA. (B) PGE1 (100 nM) inhibited the anti-proliferative effects of TIM. (C) Gene and (D) protein expression levels of the ER stress-marker proteins IRE1, ATF6 and GRP78 were increased by TIM. Data are expressed as the mean ± standard deviation (n=3). *P<0.05 and **P<0.01, vs. control group; ##P<0.01, vs. TIM alone. TIM, (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; IRE1, inositol-requiring enzyme 1; ATF6, activating transcription factor 6; PGE1, prostaglandin E1.