Figure 2.

ANXA2 association with mitophagy markers

(A) UMSCC1A cells were lysed for immunoprecipitation with LC3 (right) or species-matched IgG control antibody (left). Co-immunoprecipitation of ANXA2 is shown via western blot, and input controls were normalized to GAPDH. Images represent at least three independent experiments. Quantification of co-immunoprecipitated ANXA2 western blot bands from was normalized to uninfected, untreated control. Data are means ± SD (n = 3, ∗p < 0.05, ∗∗∗p < 0.001).

(B) Quantification of confocal images of UMSCC1A cells dual-labeled with LC3 and ANXA2 antibody, infected with P. gingivalis, and treated with LCL768. Pearson’s correlation coefficient was normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

(C) Confocal images of ceramide (green) and ANXA2 (red) in UMSCC1A cells infected with P. gingivalis and treated with LCL768 are shown.

(D) Confocal images of Atg7 (green) and ANXA2 (red) in UMSCC1A cells infected with P. gingivalis and treated with LCL768.

(E and F) Uninfected UM-SCC-1A cells were transfected with either sham or P. gingivalis FimA virulence factor proteins and treated with LCL768 or vehicle control. Confocal images of ceramide (green) and ANXA2 (red), and LC3 (green) and Tom20 (red), in uninfected cells are shown. Yellow shows colocalization. Images represent three independent experiments. Quantification of colocalization was estimated via Pearson’s correlation coefficient, completed with ImageJ Fiji software, and normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(G) Far western blotting analysis was conducted by running human recombinant ANXA2 (rANXA2) on a gel and testing direct protein-protein binding with recombinant P. gingivalis virulence factors (rFimA, rNDK). The top left blot shows direct binding between ANXA2 and FimA recombinant proteins, identified via FimA antibody. No direct binding between human recombinant ANXA2 and P. gingivalis recombinant NDK was observed in the bottom left blot. The top right blot confirms recombinant ANXA2 presence in the blot. The bottom right blot indicates that there is no cross-reactivity between recombinant ANXA2 and FimA antibody.

(H) Immunofluorescence confirmation of wildtype and FimA-deficient (ΔFimA) P. gingivalis internalization into UM-SCC-1A cells with anti-P. gingivalis antibody is shown. Quantification of mean fluorescent intensity was completed with ImageJ Fiji software and normalized to uninfected, untreated control. Values indicate mean ± SD of n = 3 independent experiments. Ns, not significant, ∗∗∗∗p < 0.0001.