Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: Eva1a knockout enhances the expression of genes implicated in fatty acid uptake. (A) RT-qPCR analysis of the relative mRNA levels of genes associated with fatty acid transport ( Cd36 , Slc27a5 , Slc27a2 , and Fabp1 ) and related transcription factor Pparg2 , lipid synthesis genes ( Fasn , Acly , Acaca , Agpat1 , Dgat2 , Mgat1 , and Scd1 ) and related transcription factor ( Srebf1 ), lipolysis and β-oxidation genes ( Pnpla2 [encoding ATGL], Lipe , Cpt1a , and Cpt2 ), and lipid assembly and secretion genes ( Apob and Mttp ) in liver tissues of Eva1a +/+ mice and Eva1a −/− mice ( n = 4). (B) Western blot analysis of fatty acid transport protein CD36, FATP5, and FATP2 in mice liver tissues ( n = 3). The blot bands were quantified by normalization to β actin by ImageJ in the lower panels. (C) Representative IHC staining image of CD36 in liver sections from mice. Scale bars: 100 μm. IHC scores of CD36 were quantified from 4 mice in the right panels. (D) Representative immunofluorescence staining image of CD36 protein (red) and nucleus (stained with DAPI, blue) in mice liver tissues ( n = 4). Scale bars: 100 μm. The right panels show the CD36 fluorescence intensity quantified using ImageJ software. (E) Comparative representative images of CD36 IHC staining in liver sections from MASLD patients and control subjects ( n = 4). Scale bars: 100 μm. IHC scores of CD36 were quantified in the right panels ( n = 4). (F) The hepatic CD36 expression from MASLD individuals and controls were analyzed by Western blot ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Immunofluorescence, Staining, Fluorescence, Software, Control

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: EVA1A knockdown up-regulates the expression of genes involved in fatty acid transport. (A) RT-qPCR analysis of the relative mRNA levels of genes associated with fatty acid intake ( CD36 , SLC27A5 , SLC27A2 , and FABP1 ) and related transcription factor PPARG2 in EVA1A-knockdown HepG2 and Huh7 cells, along with the control cells, after a 6-h treatment with 400 μM OA or left untreated. (B) Cells were treated following the protocol in panel (A) and then subjected to Western blot analysis to detect the proteins associated with fatty acid intake (CD36, FATP5, and FATP2). Protein levels were quantified in the right panels. (C) Following treatment with either 400 μM OA for 6 or 12 h or no treatment, EVA1A-knockdown cells or control cells were fixed, stained with CD36 antibody and imaged by fluorescence microscopy. Scale bars: 50 μm. Quantification of CD36 fluorescence intensity (right panels) was performed with ImageJ. (D) Assessment of CD36 expression by Western blot in EVA1A-knockdown cells or control cells under the treatment with OA for 0, 3, 6, and 12 h. Protein levels of CD36 were quantified in the right panels. * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Control, Western Blot, Staining, Fluorescence, Microscopy

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: EVA1A overexpression inhibits the expression of genes involved in fatty acid transport. (A) RT-qPCR analysis of the relative mRNA levels of fatty acid uptake genes ( CD36 , SLC27A5 , SLC27A2 , and FABP1 ) and related transcription factor PPARG2 in EVA1A-overexpressed HepG2 and Huh7 cells, along with control cells, after a 6-h treatment with 400 μM OA or left untreated. (B) Following the scheme in panel (A), cells were treated and then subjected to Western blot analysis to detect the proteins (CD36, FATP5, and FATP2). Protein levels were quantified in the right panels. (C) After 6- or 12-h treatment with 400 μM OA or left untreated, EVA1A-overexpressed cells or control cells were fixed, stained with CD36 antibody, and imaged by fluorescence microscopy. Scale bars: 50 μm. CD36 fluorescence intensity was measured with ImageJ in the right panels. (D) Western blot analysis of CD36 expression in EVA1A-overexpressing and control cells treated with 400 μM OA for the indicated times (0, 3, 6, and 12 h). Protein levels of CD36 were quantified in the right panels. * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control, Western Blot, Staining, Fluorescence, Microscopy

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: CD36 mediates the enhancement of fatty acid uptake and lipid droplet accumulation induced by EVA1A deletion. (A) EVA1A-knockdown HepG2 and Huh7 cells, along with control cells, were transfected with siNC or siCD36-1 or siCD36-2; 48 h later, cell lysates were collected and the knockdown efficiency of CD36 was assessed by Western blot. Protein levels of CD36 were quantified in the lower panels. (B) Cells were treated as (A) indicated, finally exposed to 400 μM OA for the last 6 h, then were fixed, stained with BODIPY FL-C16 (labeled fatty acids) and DAPI (labeled nucleus), and imaged by fluorescence microscopy. Scale bars: 20 μm. The fluorescence signal intensity of FL-C16 per cell was quantified by ImageJ in the lower panels ( n = 50). (C) NEFA levels in cells treated as (B) indicated. (D) Representative images of ORO staining for lipid droplets in cells treated according to the scheme in panel (B). Scale bars: 10 μm. (E) Quantitative results of cellular TG contents. * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Knockdown, Control, Transfection, Western Blot, Staining, Labeling, Fluorescence, Microscopy

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: EVA1A knockdown promotes the plasma membrane localization of CD36. (A) HepG2 and Huh7 cells with EVA1A knockdown or overexpression, along with their corresponding control cells, were treated with 400 μM OA for 0, 3, 6, and 12 h, then the cell surface CD36 was labeled with allophycocyanin (APC)-conjugated CD36 antibody and detected by flow cytometry. The further the peak of the fluorescence signal shifts to the right, the more CD36 is present on the cell membrane. (B) EVA1A knockdown or overexpression cells, along with their corresponding control cells, were subjected to confocal microscopy analysis after staining with CD36 (red) and ATP1A1 (green) antibodies. Scale bars: 5 μm. Colocalization analysis is shown in the right panel ( n = 10). (C and D) The 2 types of cells were treated as (A) indicated, then were collected, the plasma membrane fractions and cytosol fractions were extracted, and analyzed by Western blot to assess the expression of CD36, ATP1A1, and β-actin. Cell membrane CD36 levels were quantified in the lower panels. * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Knockdown, Clinical Proteomics, Membrane, Over Expression, Control, Labeling, Flow Cytometry, Fluorescence, Confocal Microscopy, Staining, Western Blot, Expressing

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: EVA1A deletion promotes the palmitoylation of CD36. (A to C) The palmitoylation levels of CD36 were assessed using the APE assay after treatment with 2-BP (50 μM, 6 h) as indicated, with or without HA. The top band indicated the palmitoylated CD36 (PEG-CD36). Detection of palmitoylated CD36 in EVA1A-knockdown HepG2 and Huh7 cells, along with control cells (A). Detection of palmitoylated CD36 in EVA1A-overexpressed HepG2 and Huh7 cells or control cells (B). Detection of palmitoylated CD36 in live tissues of Eva1a +/+ mice and Eva1a −/− mice ( n = 3) (C). Protein levels of palmitoylated CD36 were quantified in the right panels. (D to G) Relative mRNA levels of genes related to palmitoyl transferases (Z DHHC4 and Z DHHC5 ) and acyl-protein thioesterase 1 ( LYPLA1 ) in vitro (D and E) and in vivo (F and G). (H) Western blot analysis of ZDHHC5, ZDHHC4, and APT1 in EVA1A-knockdown or overexpressed HepG2 and Huh7 cells, after 6-h treatment with 400 μM OA or after being left untreated. (I) Western blot analysis of ZDHHC5, ZDHHC4, and APT1 in liver tissues of Eva1a +/+ mice and Eva1a −/− mice ( n = 3). Their protein levels were quantified in the lower panels. * P < 0.05, **P < 0.01, ***P < 0.001. HA: NH 2 OH.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Knockdown, Control, In Vitro, In Vivo, Western Blot

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: Inhibition of CD36 palmitoylation attenuates EVA1A deficiency-induced fatty acid uptake and lipid droplet accumulation. (A and E) The EVA1A-knockdown HepG2 or Huh7 cells were transfected with empty vector (pcDNA3.1) or wt-CD36, or mut-CD36 plasmid for 24 h, which had been treated with 400 μM OA during the final 6 h, then were fixed and stained with BODIPY FL-C16 (A) or ORO (E). Scale bars: 20 μm. The fluorescence signal intensity of FL-C16 per cell was quantified by ImageJ. Values were means ± SDs ( n = 50). (B) Cellular NEFA levels in cells subjected to the treatment shown in (A). (C and G) The EVA1A-knockdown cells were co-treated with 50 μM 2-BP and 400 μM OA for 6 h, then were subjected to BODIPY FL-C16 staining (C) or ORO staining (G). Scale bars: 20 μm. (D) Cellular NEFA levels in cells subjected to the treatment shown in (C). (F) Cellular TG contents in cells treated as described in (A). (H) Cellular TG contents in cells treated as described in (C). * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Inhibition, Knockdown, Transfection, Plasmid Preparation, Staining, Fluorescence

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: EVA1A facilitates CD36 mitochondrial localization and enhances β-oxidation. (A) EVA1A-overexpressing and EVA1A-knockdown HepG2 and Huh7 cells, along with their control cells, were immunostained with CD36 (red) and Tom20 (green) antibodies for confocal microscopy analysis. Scale bars: 5 μm. Colocalization analysis is shown in the right panels ( n = 10). (B) Isolation the mitochondrial fractions and isolation the fractions other than nucleus and mitochondria. Analysis of CD36, VDAC, Tom20, and β-actin expression by Western blot. Mitochondrial CD36 levels were quantified in the right panels. (C) Western blot analysis of CPT1A in EVA1A overexpression or knockdown of HepG2 and Huh7 cells, in the presence or absence of OA (400 μM, 6 h) treatment. Their protein levels were quantified in the lower panels. (D) Western blot analysis of CPT1A in liver tissues of Eva1a +/+ mice and Eva1a −/− mice. Protein levels were quantified in the lower panels ( n = 3). (E) Relative ATP production in cells. (F) Relative ATP production in liver tissues of Eva1a +/+ mice and Eva1a −/− mice. * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Knockdown, Control, Confocal Microscopy, Isolation, Expressing, Western Blot, Over Expression

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: Eva1a overexpression alleviates fatty liver in ob/ob mice. (A) Hepatic Eva1a mRNA levels measured by RT-qPCR in ob/ob mice infected with AAV- Eva1a or AAV-null ( n = 6). (B) Representative images of liver tissue from ob/ob mice stained with H&E and Oil Red O ( n = 6). Scale bars: 100 μm. (C) The body weight of ob/ob mice ( n = 6). (D) The liver index (liver-to-body weight ratios) of ob/ob mice ( n = 6). (E) The liver TG and TC levels of ob/ob mice ( n = 6). (F) Serum TG, TC, LDL-C, and HDL-C levels of ob/ob mice ( n = 6). (G) The relative CD36 and Cpt1a mRNA levels in ob/ob mice ( n = 6). (H) Determination of CD36 and CPT1A protein expression by Western blot in ob/ob mice ( n = 4). Protein levels were quantified in the right panels. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Over Expression, Quantitative RT-PCR, Infection, Staining, Expressing, Western Blot

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: The mTORC1–PPARγ2 signaling pathway mediates EVA1A deletion-induced fatty acid uptake and lipid accumulation. (A and D) Western blot analysis for PPARγ2 protein, the mTOR/P70S6K signaling pathway (p-mTORC1 and p-P70S6K), and fatty acid transport proteins CD36, FAT5, and FATP2 in EVA1A-knockdown HepG2 and Huh7 cells and control cells with or without Torin1 (300 nM, 6 h) (A) or GW9662 (5 μM, 12 h) treatment (D). Protein levels were quantified in the right panels. (B and E) Representative microscopy images of ORO staining for lipid droplets in control or EVA1A-knockdown cells with or without Torin1 (B) or GW9662 (E) treatment. Scale bars: 20 μm. (C and F) The cellular TG contents upon Torin1 treatment (C) or GW9662 treatment (F) were quantified. * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Western Blot, Knockdown, Control, Microscopy, Staining

Journal: Research

Article Title: EVA1A Regulates Hepatic Lipid Homeostasis by Modulating CD36 Expression and Its Palmitoylation

doi: 10.34133/research.1001

Figure Lengend Snippet: Schematic model of EVA1A regulating lipid metabolism in MASLD progression. Hepatic EVA1A expression levels influence MASLD progression by modulating the mTORC1–PPARγ2 signaling pathway and CD36 trafficking between the plasma membrane and mitochondria. EVA1A acts as an upstream inhibitor of mTORC1; its deletion activates the mTORC1–PPARγ2 signaling axis, up-regulating CD36 and other fatty acid transporters. In parallel, EVA1A deficiency increases the expression of palmitoyl transferases ZDHHC5 and ZDHHC4 while down-regulating the depalmitoylase APT1, which enhances CD36 palmitoylation and plasma membrane localization. This modulation enhances FFA intake, promotes lipid droplet accumulation in hepatocytes, and inhibits fatty acid β-oxidation, thus accelerating the progression of MASLD. In contrast, EVA1A overexpression inhibits mTORC1–PPARγ2 signaling axis and down-regulates CD36 and other fatty acid transporters. Meanwhile, EVA1A overexpression reduces the expression of ZDHHC5 and ZDHHC4 while up-regulating APT1 expression, promoting CD36 depalmitoylation and mitochondrial translocation. Consequently, FFA uptake and lipid droplet accumulation decrease, whereas fatty acid β-oxidation increases, which may attenuate MASLD progression. FFA, free fatty acid.

Article Snippet: Then, the slices were incubated with either an anti-EVA1A antibody (1:200, ABclonal, China) or a CD36 antibody (1:100, BOSTER or 1:500, Proteintech, China) overnight at 4 °C.

Techniques: Expressing, Clinical Proteomics, Membrane, Over Expression, Translocation Assay