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

Journal: The Journal of Experimental Medicine

Article Title: Tumor cells generate astrocyte-like cells that contribute to SHH-driven medulloblastoma relapse

doi: 10.1084/jem.20202350

Figure Lengend Snippet: BMPs induce the transdifferentiation of MB cells into astrocytes. (A and B) Primary tumor tissue ( n = 3) and relapsed tumor tissue ( n = 3) in MPG mice were collected to examine expression of BMP mRNAs by Q-PCR (A; ***, P < 0.001, Student’s t test) and phosphorylation of Smad (pSmad) by Western blotting (B). GAPDH was used as a loading control. (C) Tumor tissues from matched primary and relapsed human SHH-MB were collected to examine expression of BMP mRNAs by Q-PCR ( n = 3). ***, P < 0.001, Student’s t test. (D) Heatmap shows the expression levels of genes associated with BMP signaling in human primary MB and relapsed MB (including three paired SHH-MB: #11, #12, and #31). P < 0.05 by one-way ANOVA. (E–H) Tumor cells from MPG mice were treated with DPBS (control) or BMPs (BMP2, BMP5, or BMP7, all at 80 ng/ml) for 72 h, and then collected to examine S100β and GFP by immunocytochemistry (E). Representative images from four independent experiments are shown. The percentage of cells positive for S100β in GFP + tumor cells was quantified (F; n = 4). ***, P < 0.001 by Student’s t test. After the treatment, tumor cells were harvested to examine mRNA expression of S100β and BLBP by Q-PCR (G; ***, P < 0.001, Student’s t test) or levels of pSmad by Western blotting (H). Scale bars: 20 µm (E).

Article Snippet: Human recombinant BMP2, BMP5, and BMP7 (Peprotech) were used at 80 ng/ml for in vitro treatment of tumor cells.

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Immunocytochemistry

Journal: The Journal of Experimental Medicine

Article Title: Tumor cells generate astrocyte-like cells that contribute to SHH-driven medulloblastoma relapse

doi: 10.1084/jem.20202350

Figure Lengend Snippet: Sox9 phosphorylation mediates BMP-induced transdifferentiation of MB cells. (A–C) MB cells were virally infected with shRNA specific for Sox9 (Sh-Sox9) or scrambled shRNA (Sh-scr) for 48 h, before being harvested to examine Sox9 protein by Western blotting (A; n = 5). In addition, 24 h following the above infection, tumor cells were treated with 80 ng/ml BMP2 or DPBS for 48 h and collected to examine S100β and RFP expression by immunocytochemistry (B). Representative images from five experiments are shown. The percentage of astrocytes (S100β + , RFP + ) in infected cells (RFP + ) was quantified (C; n = 5), ***, P < 0.001 by Student’s t test. (D) MB cells were treated with 80 ng/ml BMP2, BMP5, BMP7, or DPBS in vitro for 4 h before being collected to examine Sox9 and phosphorylated Sox9 (p-Sox9) by Western blotting. Representative images from three experiments are shown. (E–G) MB cells were virally infected with GFP-tagged Sox9 S181D , wild-type Sox9 (Sox9 WT ), or an empty vector as a control. 48 h following the infection, tumor cells were harvested to examine Sox9 and p-Sox9 by Western blotting (E). In addition, tumor cells were collected to examine S100β and GFP by immunocytochemistry (F). Representative images from four experiments are shown. The percentage of astrocytes (S100β + ) among infected cells (GFP + ) was quantified (G; n = 4). ***, P < 0.001; by Student’s t test. (H) MPS mice were treated with tamoxifen (TM) or corn oil by oral gavage once a day for 7 d. MB cells were harvested to examine Sox9 protein by Western blotting. GAPDH was used a loading control. (I–K) Sox9-null MB cells were virally infected with GFP-tagged Sox9 WT , Sox9 S181A , or empty vector and treated with 80 ng/ml BMP2 or PBS for 72 h. Sox9 protein and phosphorylated Sox9 in infected MB cells were examined by Western blotting (I). Astrocytes (S100β + ) among infected cells (GFP + ) after BMP2 treatment were examined by immunocytochemistry (J). Representative images from three experiments are shown. The percentage of astrocytes (S100β + ) among infected cells (GFP + ) was quantified (K; n = 3). ***, P < 0.001 by Student’s t test. Scale bars: 20 µm (B, F, and J).

Article Snippet: Human recombinant BMP2, BMP5, and BMP7 (Peprotech) were used at 80 ng/ml for in vitro treatment of tumor cells.

Techniques: Phospho-proteomics, Infection, shRNA, Western Blot, Expressing, Immunocytochemistry, In Vitro, Plasmid Preparation, Control

Journal: The Journal of Experimental Medicine

Article Title: Tumor cells generate astrocyte-like cells that contribute to SHH-driven medulloblastoma relapse

doi: 10.1084/jem.20202350

Figure Lengend Snippet: LDN193 and LDN214 suppressed BMP signaling and Sox9 phosphorylation in MB cells. (A–C) MB cells isolated from MPG mice were treated with BMPs combined with DMSO, LDN193, or LDN214 for 48 h, before being collected to examine the expression of Id1 and Id2 by Q-PCR ( n = 4) and the levels of phosphorylation of Smad by Western blotting. Representative images from four experiments are shown. The expression of Id1 or Id2 in tumor cells after combined treatment with BMPs and LDN193 or LDN214, relative to that with the treatment of BMPs alone: BMP2 (A), BMP5 (B), and BMP7 (C). GAPDH was used as a loading control. (D–F) MB cells from MPG mice were treated with 80 ng/ml BMPs together with DMSO, LDN193, or LDN214 (1 µM) for 4 h, before being harvested to examine p-Sox9 by Western blotting. GAPDH was used for a loading control. Representative images from four experiments are shown. Levels of p-Sox9 in tumor cells treated with LDN193 or LDN214, relative to that in DMSO-treated tumor cells, were quantified ( n = 4). BMP2 (D), BMP5 (E), and BMP7 (F). **, P<0.01; ***, P < 0.001, Student’s t test.

Article Snippet: Human recombinant BMP2, BMP5, and BMP7 (Peprotech) were used at 80 ng/ml for in vitro treatment of tumor cells.

Techniques: Phospho-proteomics, Isolation, Expressing, Western Blot, Control