Journal: Molecular medicine reports

Article Title: Optimization and enrichment of induced cardiomyocytes derived from mouse fibroblasts by reprogramming with cardiac transcription factors.

doi: 10.3892/mmr.2017.8285

Figure Lengend Snippet: Figure 1. Candidate factors for cardiomyocyte induction. TTFs were transduced with individual lentiviruses expressing (A) GATA4 (B) MEF2C (C) TBX5 and (D) HAND2, and were then compared with untransduced TTFs, which were used as the control. Cells were immunostained with corresponding antibodies and DAPI. Scale bars, 100 µm. TTFs, tail‑tip fibroblasts; GATA4, GATA binding protein 4; MEF2C, myocyte‑specific enhancer factor 2C; TBX5, T‑box transcription factor 5; HAND2, heart‑ and neural crest derivatives‑expressed protein 2.

Article Snippet: The primary antibodies against GATA4 (1:100, cat. no. sc-25310), MEF2C (1:100, cat. no. sc-13268), TBX5 (1:100, cat. no. sc-376952), HAND2 (1:100, cat. no. sc-9409), α-myosin heavy chain (MHC, 1:100, cat. no. sc-20641) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), α-sarcomeric actinin (1:100, cat. no. BM0003; Boster Biological Technology, Wuhan, China) and cardiac troponin T (cTnT, 1:200, cat. no. MAB-0374; Fuzhou Maixin Biotechnology, Fuzhou, China) were applied without washing and incubated at 4 ̊C overnight.

Techniques: Transduction, Expressing, Control, Binding Assay

Journal: bioRxiv

Article Title: Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels

doi: 10.1101/2025.04.25.650540

Figure Lengend Snippet: Mef2c is sufficient to promote cone-specific gene expression and repress rod-specific gene expression in mouse. ( A ) Diagram of overexpression strategy used to test effects of MEF2C overexpression. ( B ) UMAP representation of P8 mouse retinal explants electroporated with a plasmid expressing GFP alone (Empty) or GFP in a bicistronic transcript with human MEF2C ( MEF2C ), (n = 7445 cell Empty, 11949 cells MEF2C ). Each point represents a single cell and is colored by cell type as determined by clustering and marker gene expression. ( C ) Heatmap of expression for select genes for cones, cone-like photoreceptor precursors, rod photoreceptor precursors, and rods in cells overexpressing MEF2C , scaled by gene. ( D ) Immunohistochemistry showing GFP and GNAT2 expression in P8 mouse retinas from Empty and MEF2C conditions. Scale bars, 50 µm. ( E ) Box plot of the number of Gnat2+, GFP+ cells divided by the total number of GFP+ cells (n = 8 for both conditions). P-values calculated by Wilcoxon rank-sum test. P0, postnatal day 0; P8, postnatal day 8; FACS, fluorescence-activated cell sorting; scRNA-Seq, single-cell RNA sequencing; ONL, outer nuclear layer; INL, inner nuclear layer; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; OE, overexpression.

Article Snippet: Primary antibodies utilized for staining 13LGS sections included goat anti-Otx2 polyclonal IgG (R&D Systems, BAF1979, 1:400) and rabbit anti-Mef2c polyclonal (Atlas Antibodies, HPA005533, 1:50).

Techniques: Gene Expression, Over Expression, Plasmid Preparation, Expressing, Marker, Immunohistochemistry, Fluorescence, FACS, RNA Sequencing

Journal:

Article Title: The Isopeptidase Inhibitor G5 Triggers a Caspase-independent Necrotic Death in Cells Resistant to Apoptosis

doi: 10.1074/jbc.M806113200

Figure Lengend Snippet: G5-induced necrosis is characterized by HMGB1 release. A, DKO cells, grown on coverslips, were treated for 6 h with G5, as indicated. Immunofluorescences were performed, and epifluorescence microscopy followed by deconvolution analysis was used to visualize HMGB1 localization. Hoechst 33258 staining was applied to mark nuclei. B, quantitative analysis of the immunofluorescence studies exemplified in A. WT and DKO cells, grown on coverslips were treated for 6 h with G5 as indicated, and the cytosolic localization of HMGB1 was scored after immunofluorescence analysis. C, distribution of HMGB1 among subcellular fractions. WT and DKO cells were treated for 6 h with G5 as indicated. Crude nuclear and cytosolic fractions were obtained using detergent lysis, as described under “Materials and Methods.” Protein samples were prepared for Western blotting, and membranes were probed with the indicated antibodies. The transcription factor MEF2C and tubulin were used as controls for nuclear and cytosolic fractions. D, DKO cells were treated with methyl methanesulfonate (100 μg/ml) or G5 (10 μm), culture medium was collected 20 h later, and cells were lysed in SDS buffer. HMGB1 was detected by immunoblotting in both lysates and culture medium. Histones were visualized after Coomassie Blue staining. E, electron micrograph showing a WT cell in early necrosis after incubation with 10 μm G5. F, electron micrograph showing a DKO cell in early necrosis after incubation with 10 μm G5. G, electron micrograph showing a DKO cell in late necrosis after incubation with 10 μm of G5.

Article Snippet: Western Blotting and Antibodies —Proteins obtained after an SDS denaturating lysis and sonication were transferred to a 0.2-μm pore size nitrocellulose membrane and incubated with the following antibodies: anti-caspase-2 and anti-tubulin ( 18 ); anti-caspase-3, anti-ERK1/ERK2, anti-SAPK/JNK, anti-p38 mitogen-activated protein kinase, anti-phospho-ERK1/ERK2, anti-phospho-p38 mitogen-activated protein kinase (Thr 180 /Tyr 182 ), anti-phospho-SAPK/JNK (Thr 183 /Tyr 185 ), and anti-PARP (Cell Signaling, Boston, MA); anti-HMGB1 (AbCam, Cambridge, UK); anti-HDAC4 ( 19 ); anti-LC3 ( 20 ); anti-Bcl-xL, anti-MEF2C, and anti-poly(ADP-ribose) (BD Biosciences Clontech); and anti-Bcl-2 and anti-vesicular stomatitis virus (Sigma).

Techniques: Epifluorescence Microscopy, Staining, Immunofluorescence, Lysis, Western Blot, Incubation