Fig 1: Galectin-8 exists in a complex with caspase-4 in naive cells(A) Immunoblotting for galectin-8, GBP1, NDP52, and GAPDH in the FLAG immunoprecipitates or input lysates of uninfected or S. Tm-infected IFN-γ-primed WT and LGALS8−/− HeLa cells stably expressing FLAG-galectin-8.(B) Immunoblotting for GBP1 and GAPDH in uninfected or S. Tm-infected unprimed or IFN-γ-primed WT HeLa cells.(C) Immunoblotting for GBP1 and GAPDH in the lysates of WT and LGALS8−/− HeLa cells transfected with control or GBP1 siRNAs for 48–72 h.(D and E) Cell death and IL-18 secretion by WT and LGALS8−/− HeLa cells transfected with control or GBP1 siRNAs for 48–72 h, primed with IFN-γ, and then infected with S. Tm for 6 h.(F) Immunoblotting for indicated proteins in the FLAG immunoprecipitates or input lysates of uninfected or S. Tm-infected IFN-γ-primed WT HeLa cells and LGALS8−/− HeLa cells reconstituted with FLAG-galectin-8.(G) Immunoblotting for indicated proteins in the ALFA immunoprecipitates or input lysates of uninfected or S. Tm-infected IFN-γ-primed WT HeLa cells and LGALS8−/− HeLa cells stably expressing FLAG-galectin-8 and ALFA-caspase-4.(H) Immunoblotting for indicated proteins in the ALFA immunoprecipitates or input lysates of LGALS8−/− HeLa cells reconstituted with FLAG-galectin-8 and transfected with empty pMSCV vector (EV) or pMSCV vector encoding ALFA-tagged full-length (FL), p30 enzymatic domain, or the CARD domain of caspase-4.(I) Immunoblotting for indicated proteins in the ALFA immunoprecipitates or input lysates of CASP4−/− HeLa cells reconstituted with ALFA-caspase-4 and transfected with pMSCV vector encoding FLAG-tagged FL, CRD1, or CRD2 of galectin-8.(J) STORM images of uninfected or S. Tm-infected IFN-γ-primed LGALS8−/− HeLa cells reconstituted with FLAG-galectin-8 and stained for galectin-8 (magenta), GBP-1 (cyan), or caspase-4 (cyan); white arrowheads indicate galectin-8-caspase-4 colocalization. Scale bar, 10 μm.(K) Coordinate-based colocalization analysis of STORM images showing the percentage of galectin-8 colocalizing with GBP1 or caspase-4 treated as above. Combined data from three (D and E) or data representative of three (A and F–H) or two (B, C, and I–K) experiments are shown. Data (biological replicates) are plotted in the graphs as mean ± SEM. ***p < 0.001 and ****p < 0.0001; two-way ANOVA with Sidak’s (D and E) or one-way ANOVA with Dunnett’s multiple comparison test (K). See also Figures S3 and S4.
Fig 2: Galectin-8 recruits caspase-4 to cytosol-invading bacteria. promoting LPS sensing(A) SIM images of LGALS8−/− HeLa cells stably expressing FLAG-galectin-8 and ALFA-caspase-4 infected with GFP-expressing S. Tm and stained with fluorochrome-conjugated anti-ALFA nanobody and anti-FLAG antibody to visualize caspase-4 and galectin-8, respectively. Arrows indicate colocalization of caspase-4 and galectin-8 on S. Tm. Scale bar, 500 nm.(B) Confocal images of WT and LGALS8−/− HeLa cells infected with GFP-expressing S. Tm (green) for 2 h and stained with a caspase-4 antibody (red) and phalloidin (blue) to visualize caspase-4 and the plasma membrane, respectively. White arrowheads in the boxed region show colocalization of caspase-4 with S. Tm. Scale bar, 5 μm.(C) Pearsons’s correlation coefficient for caspase-4 and S. Tm-GFP colocalization in WT and LGALS8−/− HeLa cells.(D) Percentage of intracellular S. Tm positive for caspase-4 in WT and LGALS8−/− HeLa cells treated as in (B); >200 bacteria were counted per experiment.(E and F) Representative STORM images of caspase-4 clusters in uninfected and S. Tm-infected IFN-γ-primed WT and LGALS8−/− HeLa cells stained for caspase-4 (E; magnified yellow boxed regions are shown in the bottom row; each colored dot indicates a caspase-4 cluster) and the number of caspase-4 clusters per cell is shown (F; each dot represents a cell). Scale bar, 1 μm.Combined data from two (C) or three (D) experiments or one experiment representative of two (A, E, and F) or three (B) are shown. Data (biological replicates) are plotted in the graphs as mean ± SEM. ns, not significant, *p < 0.05, and ****p < 0.0001; unpaired two-tailed t test (C and D) or two-way ANOVA with Sidak’s multiple comparison test (F). See also Figures S4 and S5.
Fig 3: Galectin-8’s role in the noncanonical inflammasome is endolysosomal-damage-sensing dependent but NDP52 independent(A) Cell death in IFN-γ-primed WT or LGALS8−/− HeLa cells pretreated with the sialidases cocktail or vehicle for 1 h and then infected with S. Tm for 6 h.(B) Intracellular bacterial loads in WT or LGALS8−/− HeLa cells pretreated with the sialidases cocktail or vehicle for 1 h and then infected with S. Tm at 2 h p.i.(C) Immunoblotting of the lysates of WT HeLa cells, LGALS8−/− HeLa cells, and LGALS8−/− HeLa cells stably expressing FLAG-tagged WT or indicated mutant galectin-8 for FLAG, galectin-8, and β-actin.(D and E) Cell death (D) and IL-18 secretion (E) by indicated stable HeLa cell lines expressing WT or various mutant galectin-8 primed with IFN-γ and infected with S. Tm at 6 h p.i.(F and G) Cell death (F) and IL-18 secretion (G) by WT and LGALS8−/− HeLa cells electroporated with PBS or LPS for 3 h.(H–J) Intracellular bacterial loads (H) and cell death (I and J) in indicated HeLa cells infected with WT or S. TmCytoKill strain for 6 h.(K and L) Cell death (K) and IL-18 (L) secretion by HeLa cells transfected with control or NDP52 siRNAs for 48–72 h, primed with IFN-γ, and then infected with S. Tm for 6 h.(M) NDP52 and β-actin in the lysates of HeLa cells transfected with control or NDP52 siRNAs for 48–72 h.Combined data from three (A, B, D–H, and J–L) or two (I) experiments or one experiment representative of three (C and M) are shown. Data (biological replicates) are plotted in the graphs as mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; two-way ANOVA with Sidak’s (B–G and I) or Tukey’s (A, H, and J–L) multiple comparison test.
Fig 4: Galectin-8 plays a role in noncanonical inflammasome activation during Salmonella infection(A) Cell death (% LDH release) and IL-18 release in wild-type (WT) and CASP4−/− HeLa cells primed with 10 ng/mL IFN-γ overnight and uninfected (medium) or infected with S. Typhimurium (S. Tm; MOI of 50 unless otherwise indicated) at 6 h post-infection (p.i.).(B–D) Cell death in HeLa cells transfected with control or indicated siRNAs for 48–72 h, primed with IFN-γ, and then infected with S. Tm for 6 h.(E) Cell death in IFN-γ-primed WT and LGALS8−/− HeLa cells infected with S. Tm at 6 h p.i.(F) Cell death in IFN-γ-primed WT and LGALS3−/− HeLa cells infected with S. Tm at 6 h p.i.(G) Cell death in HeLa cells transfected with control or indicated siRNAs for 48–72 h, primed with IFN-γ, and then infected with S. Tm for 6 h.(H) Immunoblots for full-length (FL) and N-terminal domain (NT) GSDMD in the lysates and C-terminal domain (CT) GSDMD in the supernatants of IFN-γ-primed WT and LGALS8−/− HeLa cells infected with S. Tm at 4 h p.i.(I) IL-18 in the supernatants of IFN-γ-primed WT and LGALS8−/− HeLa cells infected with S. Tm at 6 h p.i.(J and K) Cell death and IL-18 secretion by HeLa cells of indicated genotypes unprimed or primed with 10 ng/mL IFN-γ overnight and infected with S. Tm (MOIs of 50 and 100) at 6 h p.i.(L) Cell death in IFN-γ-primed WT and LGALS8−/− HeLa cells infected with WT or ΔsifA mutant S. Tm at 6 h p.i.(M and N) Cell death (M) and IL-18 secretion (N) by IFN-γ-primed WT and LGALS8−/− HeLa cells infected with S. Tm at indicated time points p.i.(O) Immunoblotting of the lysates of WT HeLa cells, LGALS8−/− HeLa cells, and LGALS8−/− HeLa cells stably expressing FLAG-galectin-8 for indicated proteins.(P and Q) Cell death (P) and IL-18 secretion (Q) by IFN-γ-primed WT HeLa cells, LGALS8−/− HeLa cells, and LGALS8−/− HeLa cells stably expressing FLAG-galectin-8 infected with S. Tm at 6 h p.i.Combined data from 2 (D, J, K, and N) or 3 (A–C, E–G, I, L, M, P, and Q) experiments or one experiment representative of two (H and O) are shown. Data (biological replicates) are plotted in the graphs as mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; two-way ANOVA with Sidak’s (A–D, G, L–N, and P) or Tukey’s (E, F, I–K, and Q) multiple comparison test. See also Figures S1 and S2.
Supplier Page from Sino Biological, Inc. for Mouse LGALS8 Gene ORF cDNA clone in cloning vector