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. 2022 Jan 12;130(1):017005. doi: 10.1289/EHP9373

Figure 7.

Figure 7A is a set of one representative images and two bar graphs. The representative images display six columns, namely, control, mono(2-ethylhexyl) phthalate, and I A C S, V L X, 3-N P, and I L-1 R A for mono(2-ethylhexyl) phthalate plus and three rows, namely, tetramethylrhodamine ethyl ester, F L C 16, and merged. The two bar graphs titled tetramethylrhodamine ethyl ester and BODIPY FL C begin subscript 16 end subscript, plotting Main stain area (micrometers squared), ranging from 0 to 80 in increments of 20 and 0 to 60 in increments of 20 (y-axis) across control, mono(2-ethylhexyl) phthalate (M), M plus I A C S, M plus V L X, M plus I L minus 1 R A (x-axis), respectively. Figure 7B is a bar graph titled Lipid droplet (L D), plotting Main stain area (micrometers squared), ranging from 0 to 50 in increments of 10 (y-axis) across control, mono(2-ethylhexyl) phthalate (M), M plus I A C S, M plus V L X, M plus I L minus 1 R A (x-axis). Figure 7C is a set of three bar graphs titled tetramethylrhodamine ethyl ester, Mito S O X, and Lipid droplet (L D), plotting Main stain area (micrometers squared), ranging from 0 to 200 in increments of 50; 0 to 70 in increments of 10; and 0 to 200 in increments of 50 (y-axis) across Hep G 2 and Co-M 2 (x-axis) for control and mono(2-ethylhexyl) phthalate. Figure 7D is a heatmap, plotting Hep G 2 control and Hep G 2 and Co-M 2 for mono(2-ethylhexyl) phthalate (columns) across Oxidative phosphorylation, including M T-A T P 6, M T-N D 2, M T-C Y B, M T-N D 4 L, A T P 6 V 0 E 2, M T-N D 1, M T-C O 1, M T-N D 5, M T-N D 6, M T-A T P 8, A T P 6 V 0 A 2 (rows). Figure 7E is a heatmap, plotting Hep G 2 and Co-M 2 for mono(2-ethylhexyl) phthalate (columns) across Glycolysis, including A L D O C, L D H A, H K 1, E N O 2, E N O 1, P K M, P G M 1, H K 2, P G K 1, T P I 1, G A P D H, P F K L, P F K P, P F K F B 4, P F K F B 3, M P I, E N O S F 1, and P F K F B 1 (rows). Figure 7F is a heatmap, plotting Hep G 2 and Co-M 2 for mono(2-ethylhexyl) phthalate (columns) across Glycerolipid metabolism, including A L D H 1 B 1, G P D 1 L, P C Y T 1 A, G P D 1, E T N K 2, P C Y T 1 B, E T N P P L, P L A 2 G 1 2 B, P L A 2 G 1 2 A, P L P P 3, P L P P 1, G P A M, A G P A T 3, and D G K K and Fatty acid metabolism, including A D H 6, A D H 4, E H H A D H, A C S L 1, H A C D 2, F A D S 1, I C M T, I D I 1, H M G C R, A C A T 2, H M G C S 1, M C A T, and F A S N (rows). A color scale depicting z score is ranging from negative 2.0 to 2.0 in increments of 2. Figure 7G is a set of two representative images. The first image displays two columns under Immunostaining of mouse liver analyzing D A P I, C L E C 4 F, I L-1 R A, and C O X 7 A 2 L. The second image displays two columns, namely, carboxymethylcellulose and diethylhexyl phthalate and two rows, namely, Wild type and Mac-K O with a scale bar of 100 micrometers.

Effect of M2 macrophages on OXPHOS and lipid metabolism in the hepatocytes. (A,B) HepG2 cells were treated with MEHP (M; 200μM) alone or in combination with IACS-10759 (IACS; 10 nM), VLX600 (VLX; 1μM), 3-nitropropanoic acid (3-NP; 2 mM), and IL-1RA (200 ng/mL) in replicate wells (n=5/treatment) for 24 h. Cells were incubated with the fluorescent dyes BODIPY FL C16 and tetramethylrhodamine ethyl ester (TMRE), or LipidTOX Neutral Lipid Stain at the end of the treatment. (A) Representative images (left) and quantification (right) of lipid uptake (FL C16) and mitochondrial membrane potential (TMRE) in HepG2 cells at the end of the treatment. (B) Quantification of lipid droplets (LDs) at the end of the treatment. The data for (A,B) are provided in Table S39. (C) Quantification of TMRE, Mitochondrial Superoxide (MitoSOX) and LD in the HepG2 cells treated with MEHP (200μM) alone or cocultured with M2 macrophages for 24h (n=5/treatment). The data are provided in Table S40. HepG2 Cells in an independent experiment with the same treatment as (C) were used for RNA sequencing (n=3 wells/treatment). Heatmaps show differently expressed genes associated with (D) OXPHOS, (E) glycolysis, and (F) lipid metabolism between different treatments. The data for (D–F) are provided in Tables S41–S43. (G) Immunofluorescence staining of CLEC4F (green), IL-1RA (yellow), and COX7A2L (red) in the liver tissues of wild-type (WT) and macrophage-specific PPARγ knockout (Mac-KO) mice treated with 0.5% (wt/vol) sodium carboxymethylcellulose (CMC; vehicle control) or DEHP (625mg/kg BW) by daily gavage for 28 d. Data for (A–C) are expressed as mean+SD. The data were analyzed using one-way (A,B) or two-way (C) ANOVA followed by Tukey’s multiple comparisons test. #p<0.05, ##p<0.01, ###p<0.001 compared with the vehicle control (CT); ***, p<0.001 compared with the MEHP group. Note: ANOVA, analysis of variance; BW, body weight; DAPI, 4′,6-diamidino-2-phenylindole; DEHP, diethylhexyl phthalate; IL, interleukin; MEHP, mono(2-ethylhexyl) phthalate; OXPHOS, oxidative phosphorylation; SD, standard deviation.