Journal: PLoS ONE

Article Title: TGF-β Blockade Reduces Mortality and Metabolic Changes in a Validated Murine Model of Pancreatic Cancer Cachexia

doi: 10.1371/journal.pone.0132786

Figure Lengend Snippet: (A) mRNA levels of Atrogin-1 and (B) MuRF1 in quadriceps muscles of mice treated with PBS or Pan02 were calculated by qPCR. (C) Similarly, levels of Atrogin-1, Myostatin, MuRF1, and β-actin in quadriceps muscle were calculated by Western blotting. (D) ZAG expression was tested in visceral adipose tissue from cohorts of mice treated with either PBS or Pan02. (E) Weight of quadriceps muscles from mouse cohorts treated with PBS or Pan02 was calculated. (F) Serum levels of MCP-1 and IL-6 were compared in PBS- and Pan02-treated mice (n = 5; *p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Protein quantification was determined by the Bradford protein assay, and samples were equally loaded on 10% polyacrylamide gels (NuPage, Grand Island, NY), electrophoresed at 200V, electrotransferred to PVDF membranes, and probed with antibodies to Atrogin-1 (1:500; ECM Biosciences, Versailles, KY), Myostatin (1:200; Abcam, Cambridge, MA), MuRF1, ZAG (1:500; both Santa Cruz Biotechnology, Dallas, TX), p21 (1:200; Santa Cruz), β-actin, Smad2, p-Smad2/3 (1:1000; all Cell Signaling, Danvers, MA), TGF-β (1:1000; Abcam), and Ezrin (1:1000; BD Transduction Laboratories, San Jose, CA).

Techniques: Muscles, Western Blot, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR

doi: 10.1038/s41392-026-02593-9

Figure Lengend Snippet: Cardiomyocyte-dependent KLF15 regulation modulates fibrosis in a cell non-autonomous manner in vivo. a Schematic showing the study design. CellChat analysis was applied to the scRNA-seq data (Dataset-3). b Scatter plot demonstrating outgoing and incoming interaction strength between the different cell types in TAC NT and Klf15 (gRNA) hearts. Macrophage (MC), endothelial cells (EC), fibroblasts (FB), pericytes (PC), and cardiomyocytes (CM). c Stacked bar plot illustrating the proportion of fibroblast subtypes (failing and non-failing) and d violin plot showing the fibroblast activation score based on the expression of Acta2, Postn, and Cadherin 11 (Cdh11) in TAC NT and Klf15 (gRNA) hearts compared to SHAM NT (gRNA). e Longitudinal section of the whole heart with Sirius red staining (scale bar = 2 mm), indicating interstitial and perivascular ventricular fibrosis in TAC NT (gRNA) compared to TAC Klf15 (gRNA) hearts and SHAM NT (gRNA). f Corresponding GO pathways analysis of the cardiomyocyte (CM) signaling towards fibroblasts (FB) based on ligand-receptor signaling enriched in each condition. g Violin plots showing the expression levels of TGF-β activation target genes Acta2 and Postn in fibroblasts of TAC NT (gRNA) and Klf15 (gRNA) hearts compared to SHAM. h Expression levels of the anti-fibrotic factor Azgp1 in cardiomyocytes of TAC NT and Klf15 (gRNA) hearts compared to SHAM NT (gRNA). i Pseudobulk expression of AZGP1 in cardiomyocytes from human hearts of non-failing donors (NF, n = 16) and patients with dilated cardiomyopathy (DCM, n = 11), and hypertrophic cardiomyopathy (HCM, n = 15). Each point represents one patient (aggregated cardiomyocyte nuclei). j Scheme illustrating the experiments of KLF15 open reading frame (ORF) overexpression (OE) fused to triple FLAG and single HA epitopes in hiPSC-derived cardiomyocytes for Chromatin Immunoprecipitation (ChIP)-seq. KLF15 ChIP-seq traces identified occupancy at the AZGP1 promoter in hiPSC-cardiomyocytes (pink traces, n = 1). IgG (black traces) served as a pulldown control. k Proof-of-concept CRISPRa KLF15 in hiPSC-cardiomyocytes. Induced expression of KLF15 compared to NT (gRNA) was validated by RT-qPCR ( n = 5-6), which resulted in a concomitant transcriptional upregulation of AZGP1 . l Increased AZGP1 secretion in CRISPRa- KLF15 (gRNA) hiPSC-cardiomyocytes as detected by ELISA compared to NT (gRNA) control and positive control AZGP1 (OE) ( n = 3-4). m Schematic of experimental setup for hiPSC-cardiomyocyte (conditions in l) and hiPSC-fibroblast crosstalk conditioned media experiment along with the RT-qPCR analysis for ACTA2 expression in hiPSC-fibroblasts upon different treatments ( n = 3 per condition, 5 days). n Representative immunofluorescence images and quantification of α-SMA expression in hiPSC-fibroblasts normalized by Vimentin expression upon treatment of the conditioned media obtained in (l). α-SMA (red), Vimentin (green), nuclei (DAPI, blue), scale bar = 50 μm. n = 6-7 field of views per condition. Mean ± 95% confidence interval (box) and minimum/maximum (whiskers) in ( i ), ( k ), and ( n ). Mean ± standard error of the mean (SEM) in ( l ) and ( m ). Wilcoxon rank sum test was used for statistical significance in ( d ), ( g ), and ( h ). DESeq2-normalized counts with genome-wide Benjamini–Hochberg correction were applied in ( i ). Student’s t -test in ( k ), One-ANOVA and Tukey’s post hoc test were used to test for statistical significance in ( l )-( n )

Article Snippet: For human recombinant AZGP1 and BMP7 protein treatment, hiPSC-fibroblasts were cultured in the fibroblast culture medium and the cells were treated with TGFβ1 (final concentration: 10 or 100 pM, Peprotech) and human recombinant AZGP1 (final concentration: 1 μg/mL, R&D Systems) or BMP7 (final concentration: 400 ng/mL, Peprotech) for 2, 24 h or 5 days with the additional Zinc 2+ supplement (final concentration: 1.5 nM, MERCK) in the medium.

Techniques: In Vivo, Activation Assay, Expressing, Staining, Over Expression, Derivative Assay, Chromatin Immunoprecipitation, ChIP-sequencing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Positive Control, Immunofluorescence, Genome Wide

Journal: Signal Transduction and Targeted Therapy

Article Title: Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR

doi: 10.1038/s41392-026-02593-9

Figure Lengend Snippet: CRISPRa-mediated transcriptional normalization of KLF15 in hiPSC-cardiomyocytes under stress. a Scheme depicting the in vitro human myocardial disease model using engineered human myocardium (EHM) with fixed metal poles (stress) for mechanical loading compared to flexible (control, CT) silicone poles representing the control condition. Representative images of EHM generated with CRISPRa hiPSC-cardiomyocytes expressing dCas9VPR (demonstrated by tdTomato, red) and NT or KLF15 (gRNA) (demonstrated by TurboGFP, green) in different conditions. Scale bar = 2 mm. b KLF15 expression in EHM measured by RT-qPCR in CT and stress EHM (corresponding wild-type hiPSC-cardiomyocyte EHM are depicted in Supplementary Fig. ) ( n = 7-15 per group from six independent hiPSC-cardiomyocyte differentiations and three independent EHM generations). c Force-Ca 2+ concentration-response curves for functional analysis of EHM comparing stress NT (gRNA) (light red); CT NT (gRNA) (light blue); stress KLF15 (gRNA) (dark red) and CT KLF15 (gRNA) (dark blue) ( n = 3-5 per group). d Immunoblot demonstrating α-SMA, ACTN2, and TurboGFP protein levels in CT/stress NT (gRNA) and CT/stress KLF15 (gRNA) and corresponding densitometry quantification showing recovery of repression upon stress KLF15 (gRNA) ( n = 3-4 per group, additional immunoblot used for quantification in Supplementary Fig. ). GAPDH was used as a loading control. e Principal component (PC) analysis and of CT NT (gRNA) ( n = 5); stress NT (gRNA) ( n = 3); and stress KLF15 (gRNA) ( n = 5) tissues subjected to bulk transcriptome analysis and f corresponding heatmap of transcript expression involved in cardiac metabolism, developmental/hypertrophic signaling, fibrosis, and structural and Ca 2+ handling genes. g Validation of a subset of genes presented in the heatmap as measured by NanoString. Selected genes ( ACTA2, BMP4 , and SLC8A1 ) that showed normalized expression upon KLF15 re-expression (further metabolic genes are presented in Supplementary Fig. ). n = 5-10 per group. h Selection of the highest significantly enriched GO pathways analysis for the indicated conditions (control NT (gRNA) versus stress NT (gRNA); stress NT (gRNA) versus stress KLF15 (gRNA)) and i enriched GO pathways analysis for NT (gRNA) versus KLF15 (gRNA) at baseline. GO pathway analysis was done based on the significantly differentially expressed genes (Log 2 FC ≥ 0.25, P -value ≤ 0.05) in each comparison for up- and downregulated genes. j Relative expression of AZGP1 in NT (gRNA) versus KLF15 (gRNA) at baseline conditions in EHM ( n = 4). k KLF15 expression measured by RT-qPCR in hiPSC-cardiomyocytes exposed to indicated concentrations of TGF-β1 ( n = 3-8, 5 days), as well as α-SMA expression evaluated by l immunoblot and m immunofluorescence in cTnT positive hiPSC-cardiomyocytes. Vehicle (Veh) exposure served as a control group. Vinculin was used as a loading control. α-SMA (red), cTnT (green), nuclei (DAPI, blue), scale bar = 50 µm. n RT-qPCR of hiPSC-cardiomyocyte with TGF-β1 exposure (final concentration: 100 pM, 5 days) showing relative KLF15 expression along with ACTN2 and de-differentiation markers ( ACTA2 and CCND1 ) ( n = 4-7 per group). Vehicle (Veh) exposure served as the control group. o Representative immunoblot images and quantification of α-SMA and ACTN2 protein expression level in hiPSC-cardiomyocytes upon treatment with indicated concentrations of TGF-β1 ( n = 3-5 per group, 5 days). Vehicle (Veh) exposure served as the control group. Vinculin was used as a loading control. p Immunoblot of downstream mediators of TGFβ signaling pathway activation, SMAD2/3, p38, and HSP27 (p-: phosphorylated) in hiPSC-cardiomyocytes upon TGF-β1 exposure (final concentration: 10 pM, 2 h, n = 3) with respective inhibitors: SB431542 (SMAD2/3 inhibitor (inhib), final concentration: 10 µM, n = 4) and SB203580 (p38 inhibitor (inhib), final concentration: 10 µM, n = 4). Vehicle only condition (Veh) was used a negative control ( n = 3). Vinculin was used as a loading control. q RT-qPCR analysis ( n = 11 - 12 per condition) of hiPSC-cardiomyocytes treated with TGF-β1 (final concentration: 10 pM, 5 days) with respective inhibitors: SB431542 (SMAD2/3 inhibitor (inh), final concentration: 10 µM) and SB203580 (p38 inhibitor (inh), final concentration: 10 µM). Vehicle (Veh) exposure served as control group. Mean ± 95% confidence interval (box) and minimum/maximum (whiskers) in ( b ), ( g ), ( j ), ( n ), and ( q ). Mean ± standard error of the mean (SEM) in ( d ), ( k ), and ( o ). Two-way ANOVA and Tukey’s post hoc test were used for ( b ), ( d ), and ( g ). Student’s t -test was used in ( j ) and ( n ). One-way ANOVA and Tukey’s post-hoc test were used to test for statistical significance in ( k ), ( o ), and ( q )

Article Snippet: For human recombinant AZGP1 and BMP7 protein treatment, hiPSC-fibroblasts were cultured in the fibroblast culture medium and the cells were treated with TGFβ1 (final concentration: 10 or 100 pM, Peprotech) and human recombinant AZGP1 (final concentration: 1 μg/mL, R&D Systems) or BMP7 (final concentration: 400 ng/mL, Peprotech) for 2, 24 h or 5 days with the additional Zinc 2+ supplement (final concentration: 1.5 nM, MERCK) in the medium.

Techniques: In Vitro, Control, Generated, Expressing, Quantitative RT-PCR, Concentration Assay, Functional Assay, Western Blot, Biomarker Discovery, Selection, Comparison, Immunofluorescence, Activation Assay, Inhibition, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR

doi: 10.1038/s41392-026-02593-9

Figure Lengend Snippet: KLF15 gene dose dependently alters cardiomyocyte de-differentiation, contractility and mitochondria network complexity. a Schematic showing the generated KLF15 genetic deletions (KO), heterozygous ( +/− ) and homozygous ( −/− ) in CRISPRa hiPSC used for cardiomyocyte differentiation, followed by treatment of control NT or KLF15 (gRNA). Representative immunofluorescence images of α-SMA and cTnT expression in KLF15 +/− and KLF15 −/− hiPSC-cardiomyocytes, along with the NT (gRNA) and KLF15 (gRNA). α-SMA (red), cTnT (gray), nuclei (DAPI, blue), scale bar = 50 µm. b Immunoblot of cardiomyocyte de-differentiation makers (α-SMA, TAGLN) in hiPSC-cardiomyocytes KLF15 +/+ , KLF15 +/− , and KLF15 −/− with the lentiviral transduction of NT or KLF15 (gRNA). Vinculin was used as a loading control. n = 2 per condition. c RT-qPCR analyses for determining KLF15 , ALDH2, and ACTA2 expression in KLF15 +/+ , KLF15 +/− , and KLF15 −/− hiPSC-cardiomyocytes transduced with NT or KLF15 (gRNA). n = 3-8 per condition. d Ca 2+ handling was assessed. Amplitudes of systolic Ca 2+ transients, rates of Ca 2+ transient upstroke velocity (time to peak), and Ca 2+ elimination kinetics (Relaxation time 50%) were assessed in KLF15 +/+ and KLF15 −/− hiPSC-cardiomyocytes. n = 30 per condition. e Mitochondria (Mito)-stress test in KLF15 +/+ , KLF15 +/− , and KLF15 −/− hiPSC-cardiomyocytes evaluated for functional measurement of mitochondrial respiration (OCR) along with quantification of ATP production, basal respiration, and maximal respiration. All the measurements were normalized by total protein level ( n = 8-12). Experiments in d and e were done in two hiPSC-cardiomyocyte differentiations. f High-resolution AiryScan confocal imaging was used to evaluate mitochondria networks in the indicated hiPSC-cardiomyocyte cultures. Mean mitochondrial volume was increased upon KLF15 activation and reduced in KLF15 +/− NT (gRNA) and both KLF15 −/− NT and KLF15 (gRNA) conditions. Branches per mitochondria were decreased in cardiomyocytes lacking KLF15 but increased upon KLF15 activation. n = 8-12 field of views per condition. g RT-qPCR assessing the levels of KLF15 expression upon TGFβ1 exposure (final concentration: 10 pM, 5 days) in hiPSC-cardiomyocytes transduced with NT and KLF15 gRNA. Vehicle condition (Veh) was used as a negative control, n = 3-5 per condition. h Ca 2+ handling evaluation and i Mito-stress were done in conditions mentioned in ( g ). Experiments in h and i were done in two hiPSC-cardiomyocyte differentiations. j RT-qPCR analyses for AZGP1 expression in KLF15 +/+ , KLF15 +/− , and KLF15 −/− hiPSC-cardiomyocytes transduced with NT or KLF15 (gRNA). n = 3-8 per condition. k Conditioned media from hiPSC-cardiomyocytes (conditions presented in 5a) were used to treat wild-type (WT) hiPSC-fibroblasts (FB) along with conditioned media from AZGP1 overexpressing (OE) hiPSC-cardiomyocytes. Quantification of α-SMA expression normalized to Vimentin is depicted. n = 4-5 field of views per condition. l Scheme showing TGFβ1 exposure (final concentration: 10 pM, 24 hours) to hiPSC-fibroblasts (FBs) with SB431542 (SMAD2/3 inhibitor (inhib), final concentration: 10 µM, n = 2), human recombinant AZGP1 protein (final concentration: 1 µg/ml, n = 2) or BMP7 protein (final concentration: 400 ng/ml, n = 2) exposure (24 h) along with immunoblot of fibrosis markers (POSTN, α-SMA and CTGF). Vehicle (Veh) exposure served as control group ( n = 2). Alpha-Tubulin was used as a loading control. m Immunoblot of downstream mediators of TGFβ signaling pathway activation, SMAD2/3, p38, AKT, and ERK1/2 (p-: phosphorylated) in hiPSC-fibroblasts with the conditions mentioned in l with 2 hours exposure time. Vinculin was used as a loading control, n = 2 per condition. n Scheme showing open reading frame overexpression of KLF15 fused to triple FLAG and one HA epitope in hiPSC-cardiomyocytes used for ChIP and Immunoprecipitation (IP) for mass spectrometry (MS) analysis. o GO pathways analysis performed on promoters of protein-coding genes identified by ChIP-seq of FLAG-HA-KLF15 compared to input showing significantly enriched GO terms. Input served as ChIP analysis control. p KLF15 ChIP-seq traces identified occupancy at the ALDH2 , ENO1 , SLC25A42 , PLORMT, and POLG2 promoter in hiPSC-cardiomyocytes (pink traces, n = 1). q Heatmap of ChIP-seq identified KLF15 target genes in EHM transduced with NT or KLF15 (gRNA) derived from bulk RNA-seq. n = 3 per condition. r Volcano plot depicting significantly enriched proteins from KLF15 IP experiments (FLAG pull-down) showing confirmation of KLF15 pull-down and identifying CSRP3 as a novel KLF15 binding partner in hiPSC-cardiomyocytes. IgG was used as a pull-down control. FC fold change. s Heatmap of known downstream targets of CSRP3 in EHM transduced with NT or KLF15 (gRNA) derived from bulk RNA-seq. n = 3 per condition. Mean ± standard error of the mean (SEM) in ( c ), ( g ), and ( j ). Mean ± 95% confidence interval (box) and minimum/maximum (whiskers) in ( d ), ( e ), ( f ), ( h ), and ( k ). Two-way ANOVA and Tukey’s post hoc test were used for ( c ), ( f ), ( g ), ( j ), and ( k ). Student’s t -test was used in ( d ). One-way ANOVA and Tukey’s post-hoc test were used to test for statistical significance in ( e ) and ( h )

Article Snippet: For human recombinant AZGP1 and BMP7 protein treatment, hiPSC-fibroblasts were cultured in the fibroblast culture medium and the cells were treated with TGFβ1 (final concentration: 10 or 100 pM, Peprotech) and human recombinant AZGP1 (final concentration: 1 μg/mL, R&D Systems) or BMP7 (final concentration: 400 ng/mL, Peprotech) for 2, 24 h or 5 days with the additional Zinc 2+ supplement (final concentration: 1.5 nM, MERCK) in the medium.

Techniques: Generated, Control, Immunofluorescence, Expressing, Western Blot, Transduction, Quantitative RT-PCR, Functional Assay, Imaging, Activation Assay, Concentration Assay, Negative Control, Inhibition, Recombinant, Over Expression, Immunoprecipitation, Mass Spectrometry, ChIP-sequencing, Derivative Assay, RNA Sequencing, Binding Assay