Journal: Scientific Reports

Article Title: FKBP51 disrupts the insulin signaling pathway and impairs mitochondrial bioenergetics in HepG2 cells

doi: 10.1038/s41598-026-40414-9

Figure Lengend Snippet: FKBP51 overexpression dampens insulin signaling in HepG2 cells but does not hinder its effects on glucose metabolism. HepG2 cells were transfected with either a plasmid coding Myc-DDK-tagged human FKBP51 (FKBP51) or an empty pCMV6 plasmid (Control) and were stimulated for 0.5 h with 100 nM insulin. ( A ) Representative blot ( n = 8), ( B ) FKBP51 protein levels, ( C ) Akt phosphorylation, ( D ) P70S6K phosphorylation, ( E ) FOXO1, ( F and G ) Glucose production assay ( n = 4), ( H ) mRNA levels (G6P, PCK1 y PDK4) ( n = 4), ( I ) Representative blot ( n = 3), GSK3β phosphorylation, ( J ) Representative imagens of glycogen synthesis assay and ( K ) Glycogen synthesis ( n = 4). One-way analysis of variance (ANOVA) followed by Tukey post hoc test. * p < 0.05 compared with control and square brackets with FKBP51 with insulin. Data are shown as means ± SEM.

Article Snippet: Proteins were resolved in SDS-polyacrylamide gels and subjected to immunoblotting overnight using antibodies specific for FKBP51 (1:1000, 12210 S, Cell Signaling, Massachusetts, USA), Akt (1:2,000, 2920 S, Cell Signaling), phospho-Ser 473 AKT (1:1000, 9271 S, Cell Signaling), phospho-p70S6K Thr389 (1:1000, 9206 S, Cell Signaling), p70S6K (1:2,000, 90205 S, Cell Signaling), phospho-FOXO1 Ser256 (1:1000, 9461 S, Cell Signaling), FOXO1 (1:1000, 14952 S, Cell Signaling), phospho-DRP1 Ser637 (1:1000, ab193216, Abcam, Cambridge, UK), DRP1 (1:1000, ab184247, Abcam), OPA1 (1:1000, ab157457, Abcam), Mfn1 (1:1000, 14739 S, Cell Signaling), Mfn2 (1:1000, 9482 S, Cell Signaling), mt-HSP70 (1:1000, MA3-D28, Invitrogen, Waltham, MA, USA), GAPDH (1:1000, 97166 S, Cell Signaling), OXPHOS (1:1000 ab110413, Abcam), phospo-GSK-3β Ser9 (1:1000, 5558, Cell Signaling) and GSK-3β (1:1,1000, 9832, Cell Signaling).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Phospho-proteomics

Journal: Scientific Reports

Article Title: FKBP51 disrupts the insulin signaling pathway and impairs mitochondrial bioenergetics in HepG2 cells

doi: 10.1038/s41598-026-40414-9

Figure Lengend Snippet: FKBP51 is partially localized in mitochondria in HepG2 cells, but does not alter mitochondrial morphology. HepG2 cells were transfected with either a plasmid coding Myc-DDK-tagged human FKBP51 (FKBP51) or an empty pCMV6 plasmid (Control) and were stimulated for 3 h with 100 nM insulin and 200 nM CCCP for 2 h. ( A ) Representative image of immunofluorescence confocal microscopy, mtHSP70 (red) for mitochondria, FKBP51 (green). Segment lines represent the cellular contours. Scale bar: 24 μm, ( B ) Quantification of mitochondria (mtHSP70)-FKBP51 colocalization using Mander’s coefficient for cell imagens, ( C ) Quantification of FKBP51-mitochondria (mtHSP70) colocalization using Mander’s coefficient for cell imagens ( n = 6), ( D ) Representative blot mitochondria-cytosol subcellular fractionation ( n = 4), ( E ) Representative image of immunofluorescence confocal microscopy MitoTracker Green FM (200 nM for 0.2 h) in live-cells, ( F ) Quantification of average mitochondrial area, ( G ) Quantification of the of mitochondria number per cell in images cells and ( H ) Mean individual mitochondrial volume of HepG2 cells ( n = 5), ( I ) Representative image of Transmission Electron Microscopy, ( J ) Quantification of mitochondrial area, ( K ) Quantification of mitochondrial perimeter, ( L ) Quantification of mitochondrial aspect ratio. One-way analysis of variance (ANOVA) followed by Tukey post hoc test. * p < 0.05 compared with control. Data are shown as means ± SEM.

Article Snippet: Proteins were resolved in SDS-polyacrylamide gels and subjected to immunoblotting overnight using antibodies specific for FKBP51 (1:1000, 12210 S, Cell Signaling, Massachusetts, USA), Akt (1:2,000, 2920 S, Cell Signaling), phospho-Ser 473 AKT (1:1000, 9271 S, Cell Signaling), phospho-p70S6K Thr389 (1:1000, 9206 S, Cell Signaling), p70S6K (1:2,000, 90205 S, Cell Signaling), phospho-FOXO1 Ser256 (1:1000, 9461 S, Cell Signaling), FOXO1 (1:1000, 14952 S, Cell Signaling), phospho-DRP1 Ser637 (1:1000, ab193216, Abcam, Cambridge, UK), DRP1 (1:1000, ab184247, Abcam), OPA1 (1:1000, ab157457, Abcam), Mfn1 (1:1000, 14739 S, Cell Signaling), Mfn2 (1:1000, 9482 S, Cell Signaling), mt-HSP70 (1:1000, MA3-D28, Invitrogen, Waltham, MA, USA), GAPDH (1:1000, 97166 S, Cell Signaling), OXPHOS (1:1000 ab110413, Abcam), phospo-GSK-3β Ser9 (1:1000, 5558, Cell Signaling) and GSK-3β (1:1,1000, 9832, Cell Signaling).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Confocal Microscopy, Fractionation, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: FKBP51 disrupts the insulin signaling pathway and impairs mitochondrial bioenergetics in HepG2 cells

doi: 10.1038/s41598-026-40414-9

Figure Lengend Snippet: FKBP51 overexpression decreases Mitofusin 2 protein levels in HepG2 cells. Cells were transfected with either a plasmid coding Myc-DDK-tagged human FKBP51 (FKBP51) or an empty pCMV6 plasmid (Control) and were stimulated for 3 h with 100 nM insulin, mtHSP70 was used as loading control. ( A ) Representative blot of proteins of mitochondrial dynamics ( n = 10), ( B ) Mitofusin 1 protein levels (MFN1), ( C ) Mitofusin 2 protein levels (MFN2), ( E ) Ratio LOPA/SOPA1, ( D ) Dynamin-related protein 1 (Drp1) phosphorylation, ( F ) Mitochondrial fission protein 1 (Fis1 protein levels), ( G ) Dynamin-related protein 1 (Drp1), protein levels, ( H ) Representative blot of mitochondrial oxidative phosphorylation chain (OXPHOS), ( I ) NADH: ubiquinone oxidoreductase subunit B8 (NDUFB8, complex I) protein level, J Succinate dehydrogenase iron-sulfur subunit (SDHB, complex II), ( K ) Ubiquinol-cytochome c reductase core protein 2 (UQCRC2, complex III), ( L ) Cytochrome c oxidase subuni1 (MTCO1, complex IV) and ( M ) ATP synthase subunit alpha (ATP5A, complex V) ( n = 8). One-way analysis of variance (ANOVA) followed by Tukey post hoc test. * p < 0.05 compared with control. Data are shown as means ± SEM.

Article Snippet: Proteins were resolved in SDS-polyacrylamide gels and subjected to immunoblotting overnight using antibodies specific for FKBP51 (1:1000, 12210 S, Cell Signaling, Massachusetts, USA), Akt (1:2,000, 2920 S, Cell Signaling), phospho-Ser 473 AKT (1:1000, 9271 S, Cell Signaling), phospho-p70S6K Thr389 (1:1000, 9206 S, Cell Signaling), p70S6K (1:2,000, 90205 S, Cell Signaling), phospho-FOXO1 Ser256 (1:1000, 9461 S, Cell Signaling), FOXO1 (1:1000, 14952 S, Cell Signaling), phospho-DRP1 Ser637 (1:1000, ab193216, Abcam, Cambridge, UK), DRP1 (1:1000, ab184247, Abcam), OPA1 (1:1000, ab157457, Abcam), Mfn1 (1:1000, 14739 S, Cell Signaling), Mfn2 (1:1000, 9482 S, Cell Signaling), mt-HSP70 (1:1000, MA3-D28, Invitrogen, Waltham, MA, USA), GAPDH (1:1000, 97166 S, Cell Signaling), OXPHOS (1:1000 ab110413, Abcam), phospo-GSK-3β Ser9 (1:1000, 5558, Cell Signaling) and GSK-3β (1:1,1000, 9832, Cell Signaling).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Phospho-proteomics

Journal: Scientific Reports

Article Title: FKBP51 disrupts the insulin signaling pathway and impairs mitochondrial bioenergetics in HepG2 cells

doi: 10.1038/s41598-026-40414-9

Figure Lengend Snippet: FKBP51 overexpression impairs mitochondrial bioenergetics. HepG2 cells were transfected with either a plasmid coding Myc-DDK-tagged human FKBP51 (FKBP51) or an empty pCMV6 plasmid (Control) and were stimulated for 3 h with 100 nM insulin. ( A ) Oxygen consumption rate (OCR) of cell, measured sequentially for 5 min, (CCCP 100 nM) ( n = 5), ( B ) Representative image of confocal microscopy of HepG2 cells loaded with TMRM then treated with CCCP 100 nM to dissipate the mitochondrial transmembrane potential. ΔB-C (ΔBasal-CCCP) was calculated as the mean fluorescence at 60 s before CCCP addition minus mean fluorescence during the last of the last 60 s of the recording, ( C ) Quantification of Δbasal-CCCP fluorescence ( n = 8), ( D ) Representative image of confocal microscopy MitoSOX 5 µM for 0.2 h with CCCP 200 nM for 1 h positive control, ( E ) Quantification of MitoSOX fluorescence ( n = 6), ( F ) Intracellular ATP levels of cells, ( G ) Graphical representation of the movement of calcium into the mitochondria by the Rhod-2 AM (4 µM for 0.2 h) probe in response to histamine 100 mM, ( H ) Area under the curve (AUC) of Ca 2+ movement to the mitochondria by the Rhod-2 AM probe in response to histamine and Slope the first 10 s in response to histamine by the Rhod-2 probe ( n = 4), ( I ) Graphical representation of the cytosolic Ca 2+ by the Fluo-4 AM (4.4 µM for 0.2 h) probe in response to histamine 100 mM, ( J ) Area under the curve (AUC) of Ca 2+ movement to the mitochondria by the Fluo-4 AM probe in response to histamine and Slope the first 10 s in response to histamine by the Fluo-4 AM probe ( n = 5). ( K ) proposed model. One-way analysis of variance (ANOVA) followed by Tukey post hoc test. * p < 0.05 compared with control and square brackets compared with FKBP51 with insulin. Data are shown as means + SEM.

Article Snippet: Proteins were resolved in SDS-polyacrylamide gels and subjected to immunoblotting overnight using antibodies specific for FKBP51 (1:1000, 12210 S, Cell Signaling, Massachusetts, USA), Akt (1:2,000, 2920 S, Cell Signaling), phospho-Ser 473 AKT (1:1000, 9271 S, Cell Signaling), phospho-p70S6K Thr389 (1:1000, 9206 S, Cell Signaling), p70S6K (1:2,000, 90205 S, Cell Signaling), phospho-FOXO1 Ser256 (1:1000, 9461 S, Cell Signaling), FOXO1 (1:1000, 14952 S, Cell Signaling), phospho-DRP1 Ser637 (1:1000, ab193216, Abcam, Cambridge, UK), DRP1 (1:1000, ab184247, Abcam), OPA1 (1:1000, ab157457, Abcam), Mfn1 (1:1000, 14739 S, Cell Signaling), Mfn2 (1:1000, 9482 S, Cell Signaling), mt-HSP70 (1:1000, MA3-D28, Invitrogen, Waltham, MA, USA), GAPDH (1:1000, 97166 S, Cell Signaling), OXPHOS (1:1000 ab110413, Abcam), phospo-GSK-3β Ser9 (1:1000, 5558, Cell Signaling) and GSK-3β (1:1,1000, 9832, Cell Signaling).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Confocal Microscopy, Fluorescence, Positive Control