Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: RNA-seq identifies ABCC5 as a potential key downstream effector of PPARγ in HS. (A) Volcano plot illustrating differentially expressed genes between the WT + HS and PPARγ-OE + HS groups. (B) GO enrichment analysis of differentially expressed genes between the WT + HS and PPARγ-OE + HS groups. (C) KEGG pathway enrichment analysis of DEGs between the WT + HS and PPARγ-OE + HS groups. (D) Heatmap displaying expression changes of ABC transporter family members across the indicated groups. (E) Measurement of cellular free fatty acids and triglycerides in cells under the indicated treatments. (F) RT-qPCR analysis of PPARγ mRNA expression in PPARγ NC + HS and PPARγ OE + HS cells. (G) RT-qPCR analysis of selected ABC transporter genes (ABCC5, ABCB1A, ABCC6, TAP2, ABCA6, ABCB4, ABCC10, ABCA2, ABCG4, ABCA1, ABCA8A, ABCA9, ABCB2, ABCB7, and ABCA3) under the specified conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the PPARγ-NC + HS group (E–G). Statistical comparisons were performed using Student's t-test (F–G) or one-way ANOVA (E).

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: Time-dependent changes in ABCC5 expression in vivo. (A) Representative immunofluorescence images of ABCC5 (green) and DAPI (blue) in cardiac tissues from sham mice and from mice subjected to HS at the indicated time points after injury. (B) Representative immunohistochemical staining of ABCC5 in cardiac tissues from sham and HS-injured mice. (C) RT-qPCR analysis of Leptin mRNA in cardiac tissues after 2.5 h or 3 weeks of heat injury. (D) Representative immunofluorescence images of ABCC5 in cardiac sections from PPARγ-cKO mice after HS). (E–F) Representative immunofluorescence images of PPARγ and ABCC5 in cardiac sections from PPARγ-cKO mice at 3 weeks after HS). Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the sham group (B–C). Statistical comparisons were performed using Student's t-test (B) or one-way ANOVA (C).

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Expressing, In Vivo, Immunofluorescence, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: ABCC5 siRNA abolishes the cardioprotective effects of PPARγ overexpression against HS ​. (A) Luciferase activity in cells co-transfected with ABCC5 wild-type or mutant (Mut1/2/3) reporter plasmids and adenovirus expressing PPARγ. (B) CUT&Tag assay using a PPARγ-specific antibody to detect PPARγ binding to the ABCC5 promoter. (C) RT-qPCR analysis of ABCC5 mRNA in cells transfected with control siRNA or ABCC5 siRNA. (D – F) Cell morphology and viability in cells transfected with ABCC5 siRNA and/or PPARγ overexpression vector under HS conditions. (G – H) Apoptosis levels measured by flow cytometry in cells transfected with ABCC5 siRNA and PPARγ-OE under HS conditions. (I – J) DCFH-DA staining for ROS detection in cells transfected with ABCC5 siRNA and PPARγ-OE under HS conditions. (K – L) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (M) Western blot analysis of PPARγ, ABCC5, ABCC1, Leptin, and β-actin (loading control) in cells treated as follows: PPARγ-NC + HS, PPARγ-OE + HS, and PPARγ-OE + ABCC5 siRNA + HS. Molecular weight markers are shown on the right. (N) Quantification of protein levels normalized to β-actin, corresponding to the blots in (M). Data are presented as mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 versus the indicated control, PPARγ + ABCC5 group (A–B), control siRNA group (C), PPARγ-NC + HS group, PPARγ-OE + HS group, or PPARγ-OE + ABCC5 siRNA + HS group (D–L), or versus the PPARγ-NC + HS group and PPARγ-OE + HS group (M − N). Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing, Binding Assay, Quantitative RT-PCR, Control, Plasmid Preparation, Flow Cytometry, Staining, Membrane, Fluorescence, Western Blot, Molecular Weight

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The PPARγ/ABCC5 pathway alleviates lipid accumulation in HS-injured mice ​. (A – D) Cardiac sections from sham mice and from mice at indicated time points after HS were stained with HE (A) , PSR (B) , Masson's trichrome (C) , or Oil Red O (D) (n = 3 per group). (E) Serum levels of HDL-C and LDL-C in sham mice and in mice 3 weeks after HS (n = 6–7 per group). Error bars represent mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the sham group. Statistical comparisons were performed using Student's t-test.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Staining

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: Rosiglitazone pretreatment alleviates HS-induced myocardial injury via the PPARγ/ABCC5 pathway in HL-1 cells ​. (A – C) Cell viability and morphology in cells treated with different concentrations of rosiglitazone (5 μM, 10 μM, 20 μM, 40 μM) under HS conditions. (D – E) Apoptosis levels in cells treated with different concentrations of rosiglitazone under HS conditions. (F–I) DHE staining (F) and DCFH-DA staining (I) for ROS detection in cells treated with different concentrations of rosiglitazone under HS conditions. (J – K) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (L) RT-qPCR analysis of PPARγ, ABCC5, Leptin, and SREBP-1c in cells treated with different concentrations of rosiglitazone under HS conditions. (M – N) Representative Western blots and quantification of PPARγ, ABCC5, ABCC1, ABCG1, ABCA1, and Leptin in cells treated with different concentrations of rosiglitazone under HS conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the control group or the HS group. Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Staining, Membrane, Fluorescence, Quantitative RT-PCR, Western Blot, Control

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The PPARγ agonist rosiglitazone confers pharmacological protection against HS-induced myocardial dysfunction ​. (A – C) Cell viability and morphology in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (D – E) Apoptosis levels measured by flow cytometry in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (F) LDH release in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (G – H) DCFH-DA staining for ROS detection in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (I – J) Mitochondrial membrane potential assessed by JC-1 fluorescence in the indicated groups. (K) RT-qPCR analysis of PPARγ and CPT1β mRNA in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. (L) Representative Western blots and quantification of PPARγ, ABCC5, PGC-1α, and PPARγ in cells transfected with PPARγ siRNA and pretreated with rosiglitazone under HS conditions. Error bars represent mean ± SD (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 versus the control group, the HS group, or the ROSI + HS group as indicated. Statistical comparisons were performed using one-way ANOVA.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques: Transfection, Flow Cytometry, Staining, Membrane, Fluorescence, Quantitative RT-PCR, Western Blot, Control

Journal: Redox Biology

Article Title: PPARγ contributes to cardioprotection against heat stroke through ABCC5-dependent lipid metabolism

doi: 10.1016/j.redox.2026.104113

Figure Lengend Snippet: The proposed scheme describing the signaling pathway of PPARγ/ABCC5-elicted cardioprotective effect against HS.

Article Snippet: For immunofluorescence, tissues and cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 h. Sections and cells were then incubated overnight at 4 °C with primary antibodies against PPARγ (Proteintech, 66936-1-1g) and ABCC5 (Bioss, bs-1437R), followed by incubation with appropriate secondary antibodies for 1 h. Images were acquired using a fluorescence microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence intensity was quantified with ImageJ Pro Plus software.

Techniques:

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: Primers used for PCR (5′-3′).

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques:

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: Functional overexpression of MRP5 in human pancreatic cancer MIA Paca-2 and PANC-1 cells. (A) High MRP5 mRNA expression in both MIA PaCa-2 and PANC-1 cells (in red colour) compared to other pancreatic cancer cell lines from the Wagner dataset stored in ONCOMINE ( https://www.oncomine.org ). (B and C) MRP5 protein detected in representative flow cytometry histogram of cell surface staining using the anti-MRP5 primary antibody (red) and isotype control IgG2a (green) on (B) MIA Paca-2 and (C) PANC-1 cells. Both the primary antibody and isotype control were labelled with Alexa Fluor 488 secondary antibody. The x-axis is the fluorescence signal intensity displayed in a linear log scale. (D) Functional expression of MRP5 detected by BCECF accumulation in MIA Paca-2 and PANC-1 cells at 15 min in the presence and absence of 50 µM benzbromarone. All data are normalised to the fluorescence intensity determined in the absence of benzbromarone. The bars represent the mean and standard deviation from three independent experiments performed in triplicates. ****P<0.0001 according to Sidak's post-hoc test that followed two-way ANOVA. MRP5, multidrug resistance protein 5.

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques: Functional Assay, Over Expression, Expressing, Flow Cytometry, Staining, Fluorescence, Standard Deviation

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: MRP5 expression at the mRNA level in (A) MIA Paca-2 and (B) PANC-1 clones transduced with scrambled control and multidrug resistance protein 5-short-hairpin RNA. Relative MRP5 mRNA expression was detected by reverse transcription-quantitative PCR. MRP5 mRNA expression was normalised to the reference gene GAPDH and relative quantitation of gene expression was calculated using the comparative threshold cycle method (2 −ΔΔCq ). All data were expressed as the mean and standard deviation from three independent experiments performed in duplicates. Cell surface protein expression of MRP5 in (C) MIA Paca-2 and (D) PANC-1 cells presented as the mean percentage of the scrambled control. The bar represents the mean and standard deviation from three independent experiments performed in triplicates. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 from Dunnett's post-hoc test that followed one-way ANOVA for comparison of all MRP5 knockdown clones to the scrambled control. (E and F) MRP5 protein detected in representative flow cytometry histograms of cell surface staining using anti-MRP5 primary antibody and Alexa Fluor 488 secondary antibody on (E) MIA Paca-2 and (F) PANC-1 clones. The x-axis is the fluorescence signal intensity displayed in a linear log scale. MRP5, multidrug resistance protein 5; shRNA, short-hairpin RNA.

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques: Expressing, Clone Assay, Transduction, shRNA, Real-time Polymerase Chain Reaction, Quantitation Assay, Standard Deviation, Comparison, Flow Cytometry, Staining, Fluorescence

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: Ligands docked on MRP5 macromolecule. Binding of (A) gemcitabine and (B) cyclic guanosine monophosphate with MRP5. MRP5 is depicted in red colour in ribbon form. MRP5, multidrug resistance protein 5.

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques: Binding Assay