Fig 2: Effect of HMGN2ac on the PYO‐mediated autophagy in the RAW 264.7 cells. The WT and KO RAW 264.7 cells were treated with 50 μM PYO or DMSO for 6 h. (A) Western blot showing LC3B II protein in the RAW 264.7 cells with or without Hmgn2 −/− upon PYO (50 μM, 6 h) or DMSO treatment, and (B) densitometric analysis showing relative expression normalized to that of the DMSO group. (C) Confocal microscopy images displaying the amount of intracellular LC3B puncta (green fluorescence, 630x) with or without Hmgn2 −/− upon PYO (50 μM, 6 h) or DMSO treatment. The nucleus was stained by DAPI (blue fluorescence, 630x), scale bar = 5 μm. (D, F) KO RAW 264.7 cells were transfected, respectively, with the GFP, 2K‐R, 3K‐R, 5K‐R and WT HMGN2 plasmids using jetPRIME for 24 h and then incubated both with 50 μM PYO and with DMSO for 6 h. The Western blot analysis showing the LC3B II protein. (E, G) Densitometric analysis showing relative expression normalized to that of the GFP plasmid. Data are expressed as mean ± SD, *P < 0.05, and n.s indicates no statistical difference, n = 3

Fig 3: HMGN2ac might have no interaction with H3K27ac. The KO RAW 264.7 cells were transfected, respectively, with GFP, 2K‐R, 3K‐R, 5K‐R and WT HMGN2 plasmids by using jetPRIME, for 24 h, and then incubated with both 50 μM PYO and DMSO for 6 h. The Western blot showing H3K27ac in RAW 264.7 cells treated both with DMSO (A) and with PYO (C), and (B, D) the densitometric analysis showing relative expression normalized by the GFP plasmid. (E) Western blot showing HMGN2 protein in the RAW 264.7 cells both with and without TSA (20 nM) upon PYO stimulation, and (F) densitometric analysis showing relative expression normalized to that of the DMSO group. (G) RT‐qPCR displaying the Hmgn2 mRNA in the RAW 264.7 cells treated with PYO (50 μM, 6 h), and DMSO is the control. (H‐I) Co‐IP showing the interaction of HMGN2 with H3K27ac. Data are expressed as mean ± SD, ***P < 0.001, and n.s indicates no statistical difference, n = 3

Fig 4: HMGN2ac and H3K27ac regulate Ulk1 transcription during autophagy. Western blot showing p‐ULK1 and ULK1 in RAW 264.7 cells (A) and PM cells (C) treated with 50 μM PYO at indicated time‐points, and (B, D) densitometric analysis showing relative expression normalized by the DMSO group. (E) RT‐qPCR analysis showing the relative Ulk1 mRNA level in the RAW 264.7 cells treated both with and without TSA (20 nM) upon PYO (50 μM, 6 h) stimulation. (F) The KO RAW 264.7 cells were transfected, respectively, with GFP, 5K‐R and WT HMGN2 plasmids by using jetPRIME for 24 h and then incubated both with 50 μM PYO and with DMSO for 6 h. RT‐qPCR showing the relative Ulk1 mRNA. (G) The schematic diagram depicting primer amplification sites in ChIP‐qPCR. TSS, transcription start site; −855 to −692, area of promoter region for primer designing. (H) ChIP‐qPCR showing the H3K27ac recruitment at the Ulk1 promoter in the RAW 264.7 cells treated both with and without TSA (20 nM) upon PYO (50 μM, 6 h) stimulation. (I, J) The KO RAW 264.7 cells were transfected respectively with GFP, 5K‐R, and WT HMGN2 plasmids by using jetPRIME for 24 h, then the cells were treated both with 50 μM PYO and with DMSO for 6 h. The ChIP‐qPCR analysis showing the HMGN2ac recruitment at the Ulk1 gene promoter. Data are expressed as mean ±SD, *P < 0.05, **P < 0.01 and ***P < 0.001, and n.s indicates no statistical difference, n = 3