Journal: Cell reports

Article Title: Fate erasure logic of gene networks underlying direct neuronal conversion of somatic cells by microRNAs

doi: 10.1016/j.celrep.2024.115153

Figure Lengend Snippet: (A) Experimental scheme to test if miR-9/9*-124 expression is sufficient to activate neuronal fate across a panel of non-neuronal cells, including dermal fibroblasts, dura fibroblasts, astrocytes, smooth muscle cells, and pericytes. (B) Representative images of pre-programmed cells across lineages (top, PID 0) or after 2 weeks of miR-9/9*-124 expression (bottom, PID 15) immunostained for neuronal marker MAP2 (gray) over DAPI (blue). Scale bars, 45 μm. (C) Quantification of MAP2-positive cells over the total number of cells (DAPI). Error bars represent SEM, dots represent the percentage of MAP2-positive cells normalized to DAPI for independent experiments ( n > 50 cells each), and numerical values represent the mean of the PID 0 cells (*** p < 0.001 and **** p < 0.0001 by paired t test). (D) qPCR quantification of pan-neuronal transcripts ( MAP2 , NEFL , NCAM2 , SYT1 ) in pre-programmed PID 0 cells across lineages or PID 15 lineages expressing miR-9/9*-124. Data represent mean ± SEM (** p < 0.01, *** p < 0.001, and **** p < 0.0001, Friedman test) and symbols represent independent reprogramming experiments, with each symbol containing data from separate wells. (E) Representative images of miNs across lineages after 2 weeks of miR-9/9*-124 expression immunostained for neuronal marker NCAM1 (green), morphological marker TUJ1 (red), and DAPI (blue). Scale bars, 150 μm. Inset is PID 0 (scale bar, 45 μm). See also .

Article Snippet: Human Dural Fibroblasts , ScienCell Research Laboratories , Cat# 1420.

Techniques: Expressing, Marker

Journal: Cell reports

Article Title: Fate erasure logic of gene networks underlying direct neuronal conversion of somatic cells by microRNAs

doi: 10.1016/j.celrep.2024.115153

Figure Lengend Snippet: (A) Schematic of miR-9/9*-124 neuronal conversion in human adult fibroblasts (HAFs). (B) Heatmap of Z scores for predicted biological upstream regulators of HAF fate erasure (HAF, PID 20 miN DEGs) and multiple lineage non-neuronal gene networks (salmon, violet, sienna3 DEG values for respective network genes) ( n = 30). (C) qPCR results for neuronal MAP2 gene expression in three low-efficiency fibroblast reprogramming lines (L5, L7, and L8). Cells were treated with CSF2 (20 ng/mL), HGF (20 ng/mL), or vehicle (H2O) for 20 days. Data are represented as mean ± SEM (n.s., not significant), and dots represent each line. (D) qPCR for neuronal MAP2 gene expression across low-efficiency reprogramming fibroblast lines (L5, L7, and L8), and dots represent each line. Cells were treated with a let-7c power inhibitor (50 nM) or non-specific inhibitor control (NS, 50 nM) for 20 days (n.s., not significant). Data are mean ± SEM. (E) qPCR results for neuronal MAP2 gene expression in low-efficiency reprogramming fibroblasts (L5, L7, and L8). Cells expressed either p53DD or a control luciferase construct for 20 days (n.s., not significant, * p < 0.05). Data are mean ± SEM, and dots show each line. (F) Representative images of moto-miNs expressing SYN-tRFP (red) with either p53DD or CTL between 1 and 3 weeks of reprogramming. Scale bars, 128 μm. (G) Time-lapse quantification of the average SYN-tRFP intensity in L1 (circle), L2 (square), L7 (triangle), and L8 (diamond) expressing p53DD or CTL between 1 and 3 weeks of reprogramming. Error bars represent SEM (multiple unpaired t tests; * p < 0.05, ** p < 0.01, and *** p < 0.001). Dots represent normalized RFP intensity images from each line, and symbols represent each line. (H) Example images of moto-miNs expressing SYN-tRFP (red) with either p53DD or CTL being patched in co-culture with hAsts from L2. (I) Summary of firing patterns observed across L1, L2, and L4 moto-miNs expressing CTL or p53DD after PID 40 ( n = 38 cells/line). (J) Representative traces of inward sodium and outward potassium currents, as well as repetitive action potential (AP) waveforms in response to current injections, with separated parallel traces on the right of each plot. Measurements derived from whole-cell current patching in L1 CTL (top) or p53DD (bottom) motomiNs (PID 40+). (K) Current (I)-voltage (V) curve for all CTL and p53DD moto-miNs recorded. Data are mean ± SEM (** p < 0.01, Wilcoxon matched-pairs signed rank test). See also and and and .

Article Snippet: Human Dural Fibroblasts , ScienCell Research Laboratories , Cat# 1420.

Techniques: Gene Expression, Control, Luciferase, Construct, Expressing, Co-Culture Assay, Derivative Assay

Journal: Cell reports

Article Title: Fate erasure logic of gene networks underlying direct neuronal conversion of somatic cells by microRNAs

doi: 10.1016/j.celrep.2024.115153

Figure Lengend Snippet:

Article Snippet: Human Dural Fibroblasts , ScienCell Research Laboratories , Cat# 1420.

Techniques: Recombinant, Electron Microscopy, Staining, Illumina Sequencing, RNA Library Preparation, Imaging, Software

Journal: PLoS ONE

Article Title: Proteomic Profiling of Human Keratinocytes Undergoing UVB-Induced Alternative Differentiation Reveals TRIpartite Motif Protein 29 as a Survival Factor

doi: 10.1371/journal.pone.0010462

Figure Lengend Snippet: A: Immunofluorescent staining of TRIM29 (green) of histological sections of reconstructed epidermis and from human skin tissue were visualized by semi-quantitative confocal microscopy. Nuclei were stained with TO-PRO-3 (blue). The results are representative of three independent experiments. B: TRIM29 mRNA abundance is more important in keratinocytes than in other cell types. Total RNA was extracted from subconfluent N-hTERT and primary keratinocytes, from dermal fibroblasts AG04431 and from hepatocarcinoma cells (HepG2). Real-time RT-PCR was performed for human TRIM29. TRIM29 mRNA abundance in fibroblasts was considered as the reference. The results (means ± SD from triplicates) (N = 3) are presented on a logarithmic scale (** p<0.01 and *** p<0.001 vs fibroblasts).

Article Snippet: Human dermal fibroblasts AG04431 (Coriell Institute for medical Research, Camden NJ, USA) were grown classically in BME (Invitrogen, Carlsbad, CA, USA) containing 10% (v:v) fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), under 5% CO 2 .

Techniques: Staining, Confocal Microscopy, Quantitative RT-PCR