Fig 2: The effect of acute ethanol-exposure on LC3-associted phagocytosis in macrophages. LC3-associated phagocytosis (LAP) in WT-BMDM and SIRT2KO-BMDM, exposed to vehicle or ethanol ± LPS. (A) Representative images of intracellular pHrodo bioparticles during phagocytosis, co-stained for LC3. (B) For each image, the co-localization of intracellular pHrodo (red) and LC3 (green) were determined as LAP and divided by the total numbers of cells (nuclei). Graph represents fluorescence quantification of LAP in WT-BMDM (n=4; * p<0.05). (C) Representative images of LAP in WT-BMDM and SIRT2KO-BMDM exposed to ethanol ± LPS. (D) Fluorescence quantification of LAP in WT-BMDM and SIRT2KO-BMDM (n=5; * p<0.05). (E) Representative images of LAP in either control siRNA or PFKP siRNA nucleo-transfected SIRT2KO-BMDM. (F) Fluorescence quantification of LAP in siRNA nucleo-transfected SIRT2KO-BMDM (n=4; * p<0.05). (G) PFKP expression was analyzed in either control siRNA or PFKP siRNA nucleo-transfected SIRT2KO-BMDM ± LPS. PFKP protein expression was detected by western blot in whole cell lysate.

Fig 3: The effect of acute ethanol-exposure-induced SIRT2 on PFKP expression in macrophages. (A) PFKP expression in WT-BMDM exposed to vehicle or ethanol ± LPS by western blot analysis. (B) PFKP western blot image quantification of PFKP protein in vehicle vs. ethanol exposed WT-BMDM, Y axis represents fold of vehicle-LPS (fold of vehicle control) (n = 4 blots; * p < 0.05). (C) PFKP expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. (D) Western blot image quantification of PFKP protein in ethanol exposed WT-BMDM and SIRT2KO-BMDM. Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05). (E) PFKP expression in Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. (F) Western blot image quantification of PFKP in AK-7 vs. DMSO treated-Ethanol-exposed WT-BMDM ± LPS, Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05).

Fig 4: SIRT2-PFKP in vivo and in vitro interaction. (A) RAW264.7 cell macrophages (RAW) ± LPS. IP of whole-cell lysates using an anti-SIRT2 antibody followed by IB analysis of PFKP and SIRT2. IP with isotype IgG control antibody was used as a negative control. (B) Western blot analysis of PFKP and SIRT2 in the whole cell lysate used as input for the SIRT2 IP. (C) In-vitro interaction between SIRT2 and PFKP using SPR. SIRT2 protein immobilized onto sensor chip and the PFKP was flowed at various concentration (15.62, 31.25, 62.5, 125, 250, 500 and 1000nM). The response units on Y axis (RU) represent quantitative assessment of protein-protein interaction. (D) wtPFKP and control plasmid transfection and IP, using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-wtPFKP and control for PFKP (turbo-GFP). HEK293T cell lysate without transfection used as a negative control. (E) Western blot analysis of control-PFKP (Turbo-GFP), wtPFKP (Turbo-GFP-wtPFKP), DDK-SIRT2 and CPA in whole cell lysate used as input for the turbo-GFP IP. (F) wtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (G) Western blot analysis of turbo-GFP wtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as an input for the TUBE IP.