Rabbit Anti-Human SQSTM1/p62 (a.a. 1-122) Polyclonal antibody

Several studies have recently revealed the regulatory mechanisms underlying female germline stem cell (FGSC) differentiation, proliferation, and apoptosis, but other biological processes such as autophagy and its mechanism in FGSCs are largely unclear. The use of small chemical compounds may be a good approach to further investigate the process and mechanism of autophagy in FGSC development. In this study, we used ZCL-082, a derivative of benzoxaboroles, to treat FGSCs. Using a cell counting kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, we found that ZCL-082 could significantly reduce the viability, proliferation, and number of FGSCs in vitro. Moreover, western blotting revealed that the expression of light chain 3 beta 2 (LC3B-II) in FGSCs was significantly increased after treatment with ZCL-082 for 3 and 6 h. Meanwhile, the expression of sequestosome-1 (SQSTM1) was significantly decreased. These results suggested that ZCL-082 can induce autophagy of FGSCs in vitro. Regarding the molecular mechanism, ZCL-082 could significantly reduce the expression of growth arrest-specific 5 (GAS5) long non-coding RNA, which could directly bind to microRNA-21a (miR-21a) and negatively regulate each other in FGSCs. Knockdown of GAS5 induced the autophagy of FGSCs, while GAS5 overexpression inhibited the autophagy of FGSCs in vitro and rescued FGSC autophagy induced by ZCL-082. Additionally, overexpression of miR-21a significantly enhanced LC3B-II protein expression while significantly reducing the expression of programmed cell death protein 4 (PDCD4) and SQSTM1 protein in FGSCs compared with control cells. The inhibition of miR-21a significantly reduced the basal or ZCL-082-induced upregulated expression of LC3B-II, and it significantly enhanced the expression of PDCD4 while downregulating the basal or ZCL-082-induced expression of SQSTM1 in FGSCs. Furthermore, the overexpression of GAS5 enhanced the protein expression of PDCD4, but knockdown of GAS5 reduced the protein expression of PDCD4. Taken together, these results suggested that ZCL-082 induced autophagy through GAS5 functioning as a competing endogenous RNA (ceRNA) sponge for miR-21a in FGSCs. It also suggested that the GAS5/miR-21a axis may be a potential therapeutic target for premature ovarian failure in the clinic.

Recent studies have begun to elucidate a role for E3 ubiquitin ligases as important mediators of the innate immune response. Our previous work defined a role for the ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) in mouse and human innate immunity. Here, we present novel data describing a role for NKLAM in regulating the immune response to Sendai virus (SeV), a murine model of paramyxoviral pneumonia. NKLAM expression was significantly upregulated by SeV infection. SeV-infected mice that are deficient in NKLAM demonstrated significantly less weight loss than wild type mice. In vivo, Sendai virus replication was attenuated in NKLAM(-/-) mice. Autophagic flux and the expression of autophagy markers LC3 and p62/SQSTM1 were also less in NKLAM(-/-) mice. Using flow cytometry, we observed less neutrophils and macrophages in the lungs of NKLAM(-/-) mice during SeV infection. Additionally, phosphorylation of STAT1 and NF kappa B p65 was lower in NKLAM(-/-) than wild type mice. The dysregulated phosphorylation profile of STAT1 and NF kappa B in NKLAM(-/-) mice correlated with decreased expression of numerous proinflammatory cytokines that are regulated by STAT1 and/or NF kappa B. The lack of NKLAM and the resulting attenuated immune response is favorable to NKLAM(-/-) mice receiving a low dose of SeV; however, at a high dose of virus, NKLAM(-/-) mice succumbed to the infection faster than wild type mice. In conclusion, our novel results indicate that NKLAM plays a role in regulating the production of pro-inflammatory cytokines during viral infection.