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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: Glia. 2018 Dec 24;67(5):802–824. doi: 10.1002/glia.23568

Figure 11. Induction of autophagy reduces ssRNA40 mediated inflammasome activity and cell death.

Figure 11

HMG cells were pre-treated with trehalose (100mM) for 6h prior to their exposure to ssRNA40 (5μg/mL). Bafilomycin A1 (Baf) co-treatment with trehalose was used to confirm the increase in autophagic flux in presence of trehalose. LyoVec and ssRNA41 (5μg/mL) treated cells were used as controls. At 24h post ssRNA40 treatment, cells were harvested and analyzed for LC3B lipidation by immunoblotting and confocal immunofluorescence microscopy. (a) Left, representative immunoblot showing expression of LC3B-II and β-Actin. Right, relative fold change (densitometric analysis) in LC3B-II protein normalized to β-Actin. Results are presented as mean ±SD, n=3; *P<0.05. (b) Representative immunofluorescent images of HMG cells treated with ssRNA40 in presence or absence of trehalose. Bafilomycin A1 co-treatment was used to confirm the increase in autophagic flux in the presence of trehalose. Vehicle (LyoVec) and ssRNA41 treated cells were used as controls. At 24h post ssRNA40 treatment cells were stained with LC3B antibody (red) and DAPI (blue). Scale bar indicates 10μm (n=3). (c and d) Trehalose (100mM) pre-treated HMG cells were exposed to ssRNA40 (5μg/mL). Vehicle (LyoVec) and ssRNA41 (5μg/mL) treated cells were used as controls. At 48h post ssRNA40 treatment, cells were harvested and lysates were analyzed for pro-IL-1β expression by immunoblotting and culture supernatants were analyzed for IL-1β and LDH release. (c) Top, representative immunoblot showing expression of pro-IL-1β. Bottom, quantitative measurement of inflammatory cytokine IL-1β release in culture supernatants from vehicle (LyoVec), ssRNA41, ssRNA40, and trehalose and ssRNA40 co-treated HMG cells by ELISA. Results are presented as mean ±SD, n=4; *P<0.05 (d) Cell death analysis by quantitative measurement of LDH release in the culture supernatants from vehicle (LyoVec), ssRNA41 (5μg/mL), ssRNA40 (5μg/mL), and trehalose (100mM) and ssRNA40 co-treated HMG cells by ELISA. Results are presented as mean ±SD, n=4; *P<0.05.

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