picrg entry vectors  (Addgene inc)

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: Cells were transfected with either a plasmid encoding Myc-DDK-tagged human FKBP51 or an empty pCMV6 plasmid (RC210608 and PS100001 , OriGene Technologies, Rockville, MD, USA), using Lipofectamine 2000® (11668019, Thermo Fisher Scientific) in opti-MEM (31985-070, Thermo Fisher Scientific) overnight.

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: Cells were transfected with either a plasmid encoding Myc-DDK-tagged human FKBP51 or an empty pCMV6 plasmid (RC210608 and PS100001 , OriGene Technologies, Rockville, MD, USA), using Lipofectamine 2000® (11668019, Thermo Fisher Scientific) in opti-MEM (31985-070, Thermo Fisher Scientific) overnight.

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: Cells were transfected with either a plasmid encoding Myc-DDK-tagged human FKBP51 or an empty pCMV6 plasmid (RC210608 and PS100001 , OriGene Technologies, Rockville, MD, USA), using Lipofectamine 2000® (11668019, Thermo Fisher Scientific) in opti-MEM (31985-070, Thermo Fisher Scientific) overnight.

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: Cells were transfected with either a plasmid encoding Myc-DDK-tagged human FKBP51 or an empty pCMV6 plasmid (RC210608 and PS100001 , OriGene Technologies, Rockville, MD, USA), using Lipofectamine 2000® (11668019, Thermo Fisher Scientific) in opti-MEM (31985-070, Thermo Fisher Scientific) overnight.

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

Journal: Cancer Research Communications

Article Title: Aberrant Hippo-YAP/TEAD Signaling Drives Malignant Transcriptional Reprogramming in External Auditory Canal Squamous Cell Carcinoma

doi: 10.1158/2767-9764.CRC-25-0626

Figure Lengend Snippet: PITX2 acts as a potential partner TF of YAP in EACSCC. A and B, Dose–response curves of VT104 in ONO2 cells examined in a proliferation assay ( n = 6; A ) and a clonogenic assay ( n = 3; B ). C, qPCR for YAP/TAZ-TEAD target genes upon treatment with VT104 (1 μmol/L) for 24 hours in ONO2 cells in vitro ( n = 3). D, YAP Co-IP experiment in ONO2 cells treated with VT104 (1 μmol/L) for 24 hours. E, Dose–response curves of VT104 in ONO2 cells upon siRNA-mediated knockdown against PITX2 ( n = 6). The IC 50 value for each group is shown. F, Proliferation assays in ONO2 cells upon siRNA-mediated knockdown against PITX2 ( n = 3). G, Transwell migration assays in ONO2 and FaDu upon siRNA-mediated knockdown against PITX2 ( n = 5). H, A volcano plot representing DEGs between PITX2-overexpressed and control ONO2 cells. Selected genes are highlighted as yellow dots. Genes with an adjusted P value < 0.01 and an absolute value of fold change >1.5 were considered significant. I, GO analysis for the DEGs upregulated in PITX2-overexpressed ONO2 cells compared with control cells in the Molecular Signature Database (MSigDB) Hallmark pathways. J, GSEA representing gene sets positively or negatively enriched in PITX2-overexpressed ONO2 cells compared with control cells in MSigDB Hallmark pathways. K, Western blots for the indicated proteins in PITX2-overexpressed and control ONO2 cells. L, Proliferation assays in PITX2-overexpressed and control ONO2 cells ( n = 5). M, Wound healing assays in PITX2-overexpressed and control ONO2 cells ( n = 3). N, Spheroid formation assays in PITX2-overexpressed and control ONO2 cells ( n = 6). O and P, Growth curves for PITX2-overexpressed or control ONO2 xenograft tumors ( O ) and bar plots for tumor weight at endpoint ( P ; n = 6). The image of tumors is also shown. Q, Representative images for HE staining as well as immunohistochemical staining for PITX2 and Ki67 in the xenograft tumors. Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: pCMV6-Entry (# PS100001 ) control plasmid and PITX2 overexpressing plasmid (#RC204179) were purchased from OriGene.

Techniques: Proliferation Assay, Clonogenic Assay, In Vitro, Co-Immunoprecipitation Assay, Knockdown, Migration, Control, Western Blot, Staining, Immunohistochemical staining

Journal: Cancer Research Communications

Article Title: Aberrant Hippo-YAP/TEAD Signaling Drives Malignant Transcriptional Reprogramming in External Auditory Canal Squamous Cell Carcinoma

doi: 10.1158/2767-9764.CRC-25-0626

Figure Lengend Snippet: YAP and PITX2 predict poor prognosis of patients with EACSCC. A, Representative images of immunohistochemical staining for YAP in EACSCC tissues. B and C, Kaplan–Meier curves for overall survival ( B ) and progression-free survival of patients with EACSCC classified according to the YAP expression levels. D, Representative images of immunohistochemical staining for PITX2 in EACSCC tissues. E and F, Kaplan–Meier curves for overall survival ( B ) and progression-free survival of patients with EACSCC classified according to the PITX2 expression levels. P values were calculated using the log-rank test.

Article Snippet: pCMV6-Entry (# PS100001 ) control plasmid and PITX2 overexpressing plasmid (#RC204179) were purchased from OriGene.

Techniques: Immunohistochemical staining, Staining, Expressing