Journal: International Journal of Biological Sciences

Article Title: DHCR24 Deficiency Drives Age-Related Meibomian Gland Dysfunction by Regulating Lipid Metabolic Imbalance and Cytosolic mtDNA-Induced cGAS-STING Activation

doi: 10.7150/ijbs.129636

Figure Lengend Snippet: DHCR24 knockdown induces mitochondrial dysfunction and activates senescence-related cGAS-STING pathway. ( A , B ) Experimental workflow for ( A ) studies in Dhcr24 -cKO mice and ( B ) in vitro experiments in DHCR24-knockdown SZ95 sebocytes. ( C ) Transmission electron microscopy (TEM) revealed mitochondrial swelling and altered cristae structure in meibomian glands (MGs) of Dhcr24 -cKO mice compared to Dhcr24 fl/fl controls. Scale bars: 10 μm. ( D , E ) Gene set enrichment analysis (GSEA) revealed significant enrichment of cGAS-STING signaling pathway in Dhcr24 -cKO mice, with ( E ) differentially expressed genes (DEGs) shown by a heatmap. ( F ) Heatmap revealed significant enrichment of DEGs associated with cellular senescence. ( G ) Western blot (WB) and relative protein levels of ( H ) cGAS, ( I ) STING, and ( J ) p-STING in Dhcr24 -cKO vs. Dhcr24 fl/fl MGs. ( K ) Representative immunofluorescence (IF) images of STING in Dhcr24 -cKO vs. Dhcr24 fl/fl MGs. Scale bars: 50 μm. ( L ) Relative DHCR24 mRNA expression in SZ95 sebocytes treated with control siRNA (siNC) and siDHCR24. ( M, N ) qPCR measurement of ( M ) cytosolic mitochondrial DNA (mtDNA) normalized to total cellular nuclear DNA (nDNA) and ( M ) cytosolic mtDNA normalized to total cellular mtDNA in SZ95 sebocytes treated with siNC and siDHCR24. ( O ) IF for Tom20 and double-stranded DNA (dsDNA) in SZ95 sebocytes treated with siNC and siDHCR24. Scale bars: 10 μm. ( P ) WB for SZ95 sebocytes treated with siNC, siDHCR24, or siDHCR24 plus the STING inhibitor H-151, and relative protein levels were quantified, including ( Q ) DHCR24, ( R ) cGAS, ( S ) STING, ( T ) p-STING, and ( U ) phospho-NF-κB p65 and NF-κB p65. ( V-Y ) Relative mRNA expression of ( V ) IFNB1, ( W ) TNF-α, ( X ) CXCL10, and ( Y ) CCL5 in SZ95 sebocytes treated with siNC, siDHCR24, or siDHCR24 plus H-151.

Article Snippet: For the treatment of STING inhibitor H-151 (Targetmol, USA; #T5674), SZ95 cells were cultured with 1 μM H-151 for 12 h.

Techniques: Knockdown, In Vitro, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Expressing, Control

Journal: bioRxiv

Article Title: Gram-positive bacteria secrete RNA aptamers to activate human STING for IL-1β release

doi: 10.1101/2021.07.21.453173

Figure Lengend Snippet: (A-D) THP1 (black), CASP1 -/- (blue), ASC -/- (grey), CASP4 -/- (green), cGAS -/- (white) and STING -/- (pattern), MΦs were infected with SA (MOI 10) and GBS (MOI 20), IL-1β production was measured in cell supernetant. (E) THP1, STING -/- , and cGAS -/- MΦs were infected with SA (MOI 10) and GBS (MOI 20), cleaved IL-1β (P17) and caspase-1 (P20) were detected in cell supernetants (Sup) and pro-IL- 1β, pro-caspase-1 and GAPDH in cell lysate by immunoblotting. (F) STING -/- (pattern), STING -/- expressing STING R232 (Black) MΦs were infected with SA (MOI 10) and GBS (MOI 20), IL-1β production was measured in cell supernetant. (G,H) Human blood derived MΦs were pre-treated with increasing concentrations of STING inhibitor (H-151) (100 µM, 50 µM, 10 µM) for 1 h and then infected with SA (MOI 10) and GBS (MOI 20) respectively. IL-1β production was measured in cell supernetant. (I) IL-1β production in wild type THP1 MΦs following transfection with increasing concentrations of SA RNA (RNA+LF) (5 µg/ml, 2.5 µg/ml, 0.1 µg/ml). (J,K,L) THP1 MΦs were pre-treated with increasing concentrations of cytosolic RNase A (RNase A+LF) (100 ng/ml, 50ng/ml, 10 ng/ml) followed by infection with SA. IL-1β production was measured in cell supernetant. Heat inactivated RNase A (100 ng/ml, 50ng/ml, 10 ng/ml) (K) and DNase I (100 ng/ml, 50ng/ml, 10 ng/ml) (L) were used as a control in similar experimental setup. Data shown are mean ±SD (n=3), representative of at least three independent experiments. Asterisks indicate statistically significant differences (∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001).

Article Snippet: Experiments with small molecule inhibitors: THP1 MΦ were pre-treated with MCC950 (5 µM, 10 µM) , Dynasore (150, 100 µM) (Enzo), KCL (60 mM, 45 mM, 75 mM), H-151 (100 µM, 50 µM, 10 µM) (ProbeChem) for 1 h prior to the stimulations.

Techniques: Infection, Western Blot, Expressing, Derivative Assay, Transfection