Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Volcano plot of DEGs in whole blood between ACLF patients (n = 17) and HCs (n = 7) in the GSE142255 dataset, with |log 2 FC| > 1 and P < 0.05. Upregulated and downregulated genes are shown in red and cyan, respectively. (B) Volcano plot of DEGs in PBMCs between 28-day non-survivors (n = 8) and survivors (n = 8) in the GSE168048 dataset, using the same thresholds as in (A). (C) Volcano plot of DEGs in liver macrophages between ACLF patients (n = 7) and HCs (n = 3) based on scRNA-seq data (PRJNA913603), with |log 2 FC| > 1 and P < 0.05. (D) Flowchart of the integrated analysis of DEGs from the three datasets. (E) Venn diagram showing the intersection of DEGs identified in analyses (A), (B), and (C). (F, G) Expression levels of PDK4 (F) and THBS1 (G) in 28-day non-survivors (n = 8) versus survivors (n = 8) in the GSE168048 cohort. Data are presented as mean ± SD. Statistical significance was assessed using an unpaired Student’s t-test. (H) ROC curves of PDK4 and THBS1 from the GSE168048 dataset for predicting 28-day mortality in ACLF patients (n = 16). (I) RT-qPCR analysis of PDK4 mRNA levels in PBMCs from ACLF patients (n = 22) and HCs (n = 6). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired Student’s t-test. (J) Correlation analysis between PDK4 expression levels in PBMCs and MELD or COSSH-ACLF II scores in patients with ACLF (n = 22). Spearman’s rank correlation coefficient was used to assess associations. (K) Multiplex immunofluorescence staining of PDK4, CD68, and CD86 in liver tissues from ACLF patients. * P < 0.05, ** P < 0.01. ACLF, acute-on-chronic liver failure; DEGs, differentially expressed genes; HCs, healthy controls; scRNA-seq, single-cell RNA sequencing; PBMCs, peripheral blood mononuclear cells; MELD, Model for End-Stage Liver Disease; COSSH-ACLF II, Chinese Group on the Study of Severe Hepatitis B-ACLF II; ROC, receiver operating characteristic; AUC, area under the curve.

Article Snippet: After deparaffinization and antigen retrieval, sections were blocked with 5% BSA and incubated with primary antibodies against PDK4 (Proteintech), CD68 (Abcam, Cambridge, UK), and CD86 (Abcam), followed by corresponding fluorophore-conjugated secondary antibodies.

Techniques: Expressing, Quantitative RT-PCR, Multiplex Assay, Immunofluorescence, Staining, RNA Sequencing

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) PDK4 mRNA expression levels in PBMCs and liver tissues from control (n = 6 per group) and ACLF (n = 6 per group) mice. (B) mRNA expression of CD86 and PDK4 in PMs isolated from control (n = 6 per group) and ACLF (n = 6 per group) mice. (C-F) CD86 and PDK4 expression in THP-1 macrophages and RAW264.7 cells after induction of M1 polarization (n = 3). Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. PDK4, pyruvate dehydrogenase kinase 4; ACLF, acute-on-chronic liver failure; PBMCs, peripheral blood mononuclear cells; PMs, peritoneal macrophages; ns, not significant.

Article Snippet: After deparaffinization and antigen retrieval, sections were blocked with 5% BSA and incubated with primary antibodies against PDK4 (Proteintech), CD68 (Abcam, Cambridge, UK), and CD86 (Abcam), followed by corresponding fluorophore-conjugated secondary antibodies.

Techniques: Expressing, Control, Isolation, Two Tailed Test

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) PDK4 expression was assessed by Western blot in THP-1 macrophages transfected with PDK4 overexpression plasmid (OE-PDK4) or empty vector (OE-EV). (B) Protein levels of the M1 markers CD86 and iNOS were analyzed by Western blot. (C) The proportion of CD86 + cells was determined by flow cytometry. (D) CD68 (red) and CD86 (green) expression was visualized by immunofluorescence staining; nuclei were counterstained with DAPI (blue); scale bar = 100 µm. (E) mRNA expression of IL-6 and TNF-α was measured by RT-qPCR. (F) Secreted levels of IL-6 and TNF-α in the supernatant were quantified by ELISA. Data are presented as mean ± SD (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ** P < 0.01, *** P < 0.001, **** P < 0.0001. OE-PDK4, PDK4 overexpression plasmid; OE-EV, overexpression empty vector; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha.

Article Snippet: After deparaffinization and antigen retrieval, sections were blocked with 5% BSA and incubated with primary antibodies against PDK4 (Proteintech), CD68 (Abcam, Cambridge, UK), and CD86 (Abcam), followed by corresponding fluorophore-conjugated secondary antibodies.

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Flow Cytometry, Immunofluorescence, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Cell viability of THP-1 macrophages treated with increasing concentrations of PDK4-IN (0–40 µM) for 24 h was assessed by CCK-8 assay. (B) Protein levels of the M1 markers CD86 and iNOS were analyzed by Western blot. (C) The proportion of CD86 + cells was determined by flow cytometry. (D) CD68 (red) and CD86 (green) expression was visualized by immunofluorescence staining; nuclei were counterstained with DAPI (blue); scale bar = 100 µm. Quantification of fluorescence intensity is shown on the right. (E) mRNA expression of IL-6 and TNF-α was measured by RT-qPCR. (F) Secreted levels of IL-6 and TNF-α in the supernatant were quantified by ELISA. (G) Flow cytometric analysis of apoptosis in LO2 cells cultured with conditioned media derived from macrophages. (H) CCK-8 assay of viability in LO2 cells cultured with conditioned media derived from macrophages. Data are presented as mean ± SD (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. PDK4-IN, pyruvate dehydrogenase kinase 4 inhibitor; CCK-8, Cell Counting Kit-8; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

Article Snippet: After deparaffinization and antigen retrieval, sections were blocked with 5% BSA and incubated with primary antibodies against PDK4 (Proteintech), CD68 (Abcam, Cambridge, UK), and CD86 (Abcam), followed by corresponding fluorophore-conjugated secondary antibodies.

Techniques: CCK-8 Assay, Western Blot, Flow Cytometry, Expressing, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay, Two Tailed Test, Cell Counting

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Volcano plot showing DEGs in macrophages stimulated with LPS/IFN-γ, with or without pretreatment with PDK4-IN. Upregulated genes are shown in red, downregulated genes in cyan. (B) GSEA of transcriptomic data. (C, D) Western blot analysis and quantification of total STAT1 and p-STAT1. Cells were pretreated with either PDK4-IN or PDK4 overexpression vector, followed by LPS/IFN-γ stimulation. (E) Western blot analysis and quantification of M1 polarization markers CD86 and iNOS under PDK4 overexpression in the presence or absence of the STAT1 inhibitor fludarabine. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. +, with; -. without; PDK4, pyruvate dehydrogenase kinase 4; STAT1, signal transducer and activator of transcription 1; p-STAT1, phosphorylated STAT1; DEGs, differentially expressed genes; GSEA, gene set enrichment analysis; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase.

Article Snippet: After deparaffinization and antigen retrieval, sections were blocked with 5% BSA and incubated with primary antibodies against PDK4 (Proteintech), CD68 (Abcam, Cambridge, UK), and CD86 (Abcam), followed by corresponding fluorophore-conjugated secondary antibodies.

Techniques: Western Blot, Over Expression, Plasmid Preparation

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Schematic diagram of the experimental protocol. (B) Gross morphology and HE staining of liver tissues from control, ACLF, and PDK4-IN groups (n = 6 per group). Scale bars: 1 cm for gross morphology and 50 μm for HE-stained images. (C) Serum levels of ALT, AST, and TBIL. (D) Serum concentrations of TNF-α, IL-1β, and IL-6. (E) Immunohistochemical staining for CD86 in liver tissues. Scale bar = 50 µm. (F, G) Western blot and RT-qPCR analyses of CD86 and iNOS protein and mRNA expression levels in liver tissues. (H) RT-qPCR analysis of CD86, iNOS, and IL-6 mRNA levels in PMs. Data are presented as mean ± SD (n = 6 per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ACLF, acute-on-chronic liver failure; PDK4, pyruvate dehydrogenase kinase 4; PDK4-IN, PDK4 inhibitor; PMs, peritoneal macrophages; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase.

Article Snippet: After deparaffinization and antigen retrieval, sections were blocked with 5% BSA and incubated with primary antibodies against PDK4 (Proteintech), CD68 (Abcam, Cambridge, UK), and CD86 (Abcam), followed by corresponding fluorophore-conjugated secondary antibodies.

Techniques: Staining, Control, Immunohistochemical staining, Western Blot, Quantitative RT-PCR, Expressing

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Volcano plot of DEGs in whole blood between ACLF patients (n = 17) and HCs (n = 7) in the GSE142255 dataset, with |log 2 FC| > 1 and P < 0.05. Upregulated and downregulated genes are shown in red and cyan, respectively. (B) Volcano plot of DEGs in PBMCs between 28-day non-survivors (n = 8) and survivors (n = 8) in the GSE168048 dataset, using the same thresholds as in (A). (C) Volcano plot of DEGs in liver macrophages between ACLF patients (n = 7) and HCs (n = 3) based on scRNA-seq data (PRJNA913603), with |log 2 FC| > 1 and P < 0.05. (D) Flowchart of the integrated analysis of DEGs from the three datasets. (E) Venn diagram showing the intersection of DEGs identified in analyses (A), (B), and (C). (F, G) Expression levels of PDK4 (F) and THBS1 (G) in 28-day non-survivors (n = 8) versus survivors (n = 8) in the GSE168048 cohort. Data are presented as mean ± SD. Statistical significance was assessed using an unpaired Student’s t-test. (H) ROC curves of PDK4 and THBS1 from the GSE168048 dataset for predicting 28-day mortality in ACLF patients (n = 16). (I) RT-qPCR analysis of PDK4 mRNA levels in PBMCs from ACLF patients (n = 22) and HCs (n = 6). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired Student’s t-test. (J) Correlation analysis between PDK4 expression levels in PBMCs and MELD or COSSH-ACLF II scores in patients with ACLF (n = 22). Spearman’s rank correlation coefficient was used to assess associations. (K) Multiplex immunofluorescence staining of PDK4, CD68, and CD86 in liver tissues from ACLF patients. * P < 0.05, ** P < 0.01. ACLF, acute-on-chronic liver failure; DEGs, differentially expressed genes; HCs, healthy controls; scRNA-seq, single-cell RNA sequencing; PBMCs, peripheral blood mononuclear cells; MELD, Model for End-Stage Liver Disease; COSSH-ACLF II, Chinese Group on the Study of Severe Hepatitis B-ACLF II; ROC, receiver operating characteristic; AUC, area under the curve.

Article Snippet: PDK4-IN group: same treatment as the ACLF group, plus oral administration of PDK4-IN-1 hydrochloride (PDK4-IN, 50 mg/kg, TargetMol, Shanghai, China) 8 h before LPS/D-GalN.

Techniques: Expressing, Quantitative RT-PCR, Multiplex Assay, Immunofluorescence, Staining, RNA Sequencing

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) PDK4 mRNA expression levels in PBMCs and liver tissues from control (n = 6 per group) and ACLF (n = 6 per group) mice. (B) mRNA expression of CD86 and PDK4 in PMs isolated from control (n = 6 per group) and ACLF (n = 6 per group) mice. (C-F) CD86 and PDK4 expression in THP-1 macrophages and RAW264.7 cells after induction of M1 polarization (n = 3). Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. PDK4, pyruvate dehydrogenase kinase 4; ACLF, acute-on-chronic liver failure; PBMCs, peripheral blood mononuclear cells; PMs, peritoneal macrophages; ns, not significant.

Article Snippet: PDK4-IN group: same treatment as the ACLF group, plus oral administration of PDK4-IN-1 hydrochloride (PDK4-IN, 50 mg/kg, TargetMol, Shanghai, China) 8 h before LPS/D-GalN.

Techniques: Expressing, Control, Isolation, Two Tailed Test

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) PDK4 expression was assessed by Western blot in THP-1 macrophages transfected with PDK4 overexpression plasmid (OE-PDK4) or empty vector (OE-EV). (B) Protein levels of the M1 markers CD86 and iNOS were analyzed by Western blot. (C) The proportion of CD86 + cells was determined by flow cytometry. (D) CD68 (red) and CD86 (green) expression was visualized by immunofluorescence staining; nuclei were counterstained with DAPI (blue); scale bar = 100 µm. (E) mRNA expression of IL-6 and TNF-α was measured by RT-qPCR. (F) Secreted levels of IL-6 and TNF-α in the supernatant were quantified by ELISA. Data are presented as mean ± SD (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ** P < 0.01, *** P < 0.001, **** P < 0.0001. OE-PDK4, PDK4 overexpression plasmid; OE-EV, overexpression empty vector; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha.

Article Snippet: PDK4-IN group: same treatment as the ACLF group, plus oral administration of PDK4-IN-1 hydrochloride (PDK4-IN, 50 mg/kg, TargetMol, Shanghai, China) 8 h before LPS/D-GalN.

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Flow Cytometry, Immunofluorescence, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Cell viability of THP-1 macrophages treated with increasing concentrations of PDK4-IN (0–40 µM) for 24 h was assessed by CCK-8 assay. (B) Protein levels of the M1 markers CD86 and iNOS were analyzed by Western blot. (C) The proportion of CD86 + cells was determined by flow cytometry. (D) CD68 (red) and CD86 (green) expression was visualized by immunofluorescence staining; nuclei were counterstained with DAPI (blue); scale bar = 100 µm. Quantification of fluorescence intensity is shown on the right. (E) mRNA expression of IL-6 and TNF-α was measured by RT-qPCR. (F) Secreted levels of IL-6 and TNF-α in the supernatant were quantified by ELISA. (G) Flow cytometric analysis of apoptosis in LO2 cells cultured with conditioned media derived from macrophages. (H) CCK-8 assay of viability in LO2 cells cultured with conditioned media derived from macrophages. Data are presented as mean ± SD (n = 3). Statistical significance was determined by unpaired two-tailed Student’s t-test (for comparisons between two groups) or one-way ANOVA (for comparisons among multiple groups). ns, P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. PDK4-IN, pyruvate dehydrogenase kinase 4 inhibitor; CCK-8, Cell Counting Kit-8; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

Article Snippet: PDK4-IN group: same treatment as the ACLF group, plus oral administration of PDK4-IN-1 hydrochloride (PDK4-IN, 50 mg/kg, TargetMol, Shanghai, China) 8 h before LPS/D-GalN.

Techniques: CCK-8 Assay, Western Blot, Flow Cytometry, Expressing, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay, Two Tailed Test, Cell Counting

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Volcano plot showing DEGs in macrophages stimulated with LPS/IFN-γ, with or without pretreatment with PDK4-IN. Upregulated genes are shown in red, downregulated genes in cyan. (B) GSEA of transcriptomic data. (C, D) Western blot analysis and quantification of total STAT1 and p-STAT1. Cells were pretreated with either PDK4-IN or PDK4 overexpression vector, followed by LPS/IFN-γ stimulation. (E) Western blot analysis and quantification of M1 polarization markers CD86 and iNOS under PDK4 overexpression in the presence or absence of the STAT1 inhibitor fludarabine. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. +, with; -. without; PDK4, pyruvate dehydrogenase kinase 4; STAT1, signal transducer and activator of transcription 1; p-STAT1, phosphorylated STAT1; DEGs, differentially expressed genes; GSEA, gene set enrichment analysis; LPS, lipopolysaccharide; IFN-γ, interferon-gamma; CD86, cluster of differentiation 86; iNOS, inducible nitric oxide synthase.

Article Snippet: PDK4-IN group: same treatment as the ACLF group, plus oral administration of PDK4-IN-1 hydrochloride (PDK4-IN, 50 mg/kg, TargetMol, Shanghai, China) 8 h before LPS/D-GalN.

Techniques: Western Blot, Over Expression, Plasmid Preparation

Journal: Journal of Clinical and Translational Hepatology

Article Title: PDK4 Regulates Inflammatory Injury in Acute-on-chronic Liver Failure by Phosphorylating STAT1-mediated M1 Polarization of Macrophages

doi: 10.14218/JCTH.2025.00343

Figure Lengend Snippet: (A) Schematic diagram of the experimental protocol. (B) Gross morphology and HE staining of liver tissues from control, ACLF, and PDK4-IN groups (n = 6 per group). Scale bars: 1 cm for gross morphology and 50 μm for HE-stained images. (C) Serum levels of ALT, AST, and TBIL. (D) Serum concentrations of TNF-α, IL-1β, and IL-6. (E) Immunohistochemical staining for CD86 in liver tissues. Scale bar = 50 µm. (F, G) Western blot and RT-qPCR analyses of CD86 and iNOS protein and mRNA expression levels in liver tissues. (H) RT-qPCR analysis of CD86, iNOS, and IL-6 mRNA levels in PMs. Data are presented as mean ± SD (n = 6 per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ACLF, acute-on-chronic liver failure; PDK4, pyruvate dehydrogenase kinase 4; PDK4-IN, PDK4 inhibitor; PMs, peritoneal macrophages; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase.

Article Snippet: PDK4-IN group: same treatment as the ACLF group, plus oral administration of PDK4-IN-1 hydrochloride (PDK4-IN, 50 mg/kg, TargetMol, Shanghai, China) 8 h before LPS/D-GalN.

Techniques: Staining, Control, Immunohistochemical staining, Western Blot, Quantitative RT-PCR, Expressing

Journal: Metabolic Engineering Communications

Article Title: Substrate stiffness-dependent metabolic reprogramming of iPSC-derived cardiomyocytes on physiological PDMS polymers

doi: 10.1016/j.mec.2025.e00266

Figure Lengend Snippet: qPCR transcript expression of metabolic genes in day 25 iPSC-CMs cultured on 20 kPa, 130 kPa PDMS and plastic. Transcript levels of (A) PPARδ (B) PPARα (C) PPARγ (D) PDK4 (E) CD36 (F) CPT1B (G) HK2 (H) PFKM . Data has been normalised to TBP and made relative to plastic. Values are presented as mean ± SD. Each data point refers to 1 well of cells plated as 3 technical replicates, with a total of n = 5 batches of differentiated iPSC-CMs per condition. Statistical significance was assessed with a Kruskal-Wallis test as data was not normally distributed, with p values determined as <0.01 (∗∗).

Article Snippet: Primary antibodies, rabbit PPARδ (PA1-823A, Invitrogen) at 1:750, rabbit PDK4 (ProteinTech,12949-1-AP) at 1:1000 and mouse GAPDH (Cell Signalling Technology, D4C6R) at 1:1000 were diluted in 5 % milk TBST and left overnight at 4 °C on a shaker.

Techniques: Expressing, Cell Culture

Journal: Metabolic Engineering Communications

Article Title: Substrate stiffness-dependent metabolic reprogramming of iPSC-derived cardiomyocytes on physiological PDMS polymers

doi: 10.1016/j.mec.2025.e00266

Figure Lengend Snippet: Protein expression of metabolic genes. (A) Western blot of PPARδ, PDK4 and GAPDH (B) Quantification of PPARδ expression. (C) Quantification of PDK4 expression. Data has been normalised to GAPDH and made relative to plastic. Values are presented as mean ± SD. Each data point refers to 1 well of cells per condition, across a total of n = 4 batches of iPSC-CM differentiations. Statistical significance was assessed with a Kruskal-Wallis test as data was not normally distributed, with no significant differences observed.

Article Snippet: Primary antibodies, rabbit PPARδ (PA1-823A, Invitrogen) at 1:750, rabbit PDK4 (ProteinTech,12949-1-AP) at 1:1000 and mouse GAPDH (Cell Signalling Technology, D4C6R) at 1:1000 were diluted in 5 % milk TBST and left overnight at 4 °C on a shaker.

Techniques: Expressing, Western Blot