Journal: Cell discovery

Article Title: RhoA-mediated G 12 -G 13 signaling maintains muscle stem cell quiescence and prevents stem cell loss.

doi: 10.1038/s41421-024-00696-7

Figure Lengend Snippet: Fig. 2 Pro-quiescence effects of ET-3 and NT on MuSCs are mediated by ETB and NTS2. a RT-qPCR analysis of Ednrb, Ntsr1, and Ntsr2 expression in freshly isolated MuSCs (FSC) and activated stem cells (ASC) (n = 3). b Representative images of immunostaining for ETB/NTS1/ NTS2 (red), PAX7 (green) and DAPI (blue) on FDB myofibers of WT mice at 0 h (upper panel) and 24 h (lower panel) of culturing. c The quantification of GPCR staining intensity (n = 3). d, e Quantification of the ratios of PAX7+KI67+ (d) and PAX7+MYOD‒ (e) MuSCs on FDB myofibers after 24 h culturing in the presence of DMSO, BQ788 or SR142948A (n = 6). f, g Quantification of the ratio of PAX7+KI67+ (f) and PAX7+MYOD−(g) MuSCs on FDB myofibers after 24 h culturing in the presence of DMSO, ET-3, NT, ET-3 + BQ788, NT + SR142948A, or ET- 3 + NT (n = 6). h‒l Representative images (h) and quantification of PAX7+ (i), the ratio of PAX7+KI67+ (j), and PAX7+MYOD+ (k) cells, and quantification of MuSCs outside the basal limina (l) on transverse sections of WT mice TA muscle after intramuscular injection of solvent, BQ788 (1 mg/kg bodyweight), or SR142948A (0.5 mg/kg bodyweight), respectively (n = 3). The cartoon in h depicts the outline of the experimental design. The data represent means ± SEM, analyzed by unpaired t-test (a, c) and one-way ANOVA with Bonferroni’s multiple comparisons test (d‒g and i‒l). ND not detected. Scale bars: 5 µm in b, 10 µm in h.

Article Snippet: The whole length cDNA of human EDNRB and NTSR2 genes were cloned from EDNRBTango (# 66458, Addgene) and NTSR2-Tango (# 66458, Addgene) plasmids, respectively, and inserted into the pcDNA3.1(+) vector subsequently.

Techniques: Quantitative RT-PCR, Expressing, Isolation, Immunostaining, Staining, Injection, Solvent

Journal: bioRxiv

Article Title: Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 with functional confirmation

doi: 10.1101/2024.04.02.587857

Figure Lengend Snippet: A . Representative immunofluorescent images of GFP reporter tag expression in the CRISPR-Cas9 CACNA1E KO plasmid treated cells (bottom), compared to the negative control cells (top). Representative brightfield images (left) and the fluorescent images (right) were taken using a Biorad ZOE Fluorescent cell imager. Scale bar 100μm. B . Percent transfection efficiency for the CACNA1E CRISPR plasmid quantified by fluorescent tag expression in neurons alone and in the entire hDRG culture. C . Percent transfection efficiency for the NTSR2 CRISPR plasmid quantified by fluorescent tag expression in neurons alone and in the entire hDRG culture. (Images not shown). D . CellTiter Glo 2.0 Assay demonstrating viability of cells may differ between the cells exposed to plasmids containing the GFP compared to the mCherry reporter. Both NTSR2 and CACNA1E DNA plasmids with GFP reporter tags show a significant decrease in viability compared to non-treated control cells but not in comparison to lipofectamine treated NC cells. Both lipofectamine alone and the plasmid containing the mCherry tag ( TRPV1 ) show no significant decrease in viability compared to the non-treated control cells. Luminescence was measured across two different donors with 3 individual replicates per treatment group using a Tecan Spark 20 plate reader; *** = p < 0.001; ** = p < 0.01; by 1-Way ANOVA with Tukey’s post hoc test E . Representative Western blot and quantification of NTSR2; data was normalized to GAPDH, then further normalized to the negative control group (100%). ** = p < 0.01; * = p < 0.1 by unpaired 2-tailed t - test. F . Representative Western blot and quantification of Cav2.3; data was normalized to GAPDH, then further normalized to the negative control group (100%). * = p < 0.05 by unpaired 2-tailed t -test. G . Representative image of DNA gel demonstrating multiple DNA band fragments in the NTSR2 & CACNA1E CRISPR plasmid exposed sample compared to the single negative control sample band.

Article Snippet: Antibodies used were as follows: Peripherin (chicken 1:1000; EnCor, AB_2284443) Cav2.3 (Rabbit, Alomone labs, 1:100 AB_2039777; lot ACC006AN0302), NTSR2 (rabbit, 1:200, ThermoFisher BS-12004R), TRPV1 (Rabbit, 1:500 Fisher scientific PA1748) GAPDH (mouse, Cell signaling 97166s) secondary Alexa Fluor goat anti-mouse immunoglobulin G (IgG) 594 (1:5000; Invitrogen, A11032, lot 1985396), and secondary Alexa Fluor goat anti-mouse IgG 488 (1:5000; Invitrogen A-11034, lot 2110499).

Techniques: Expressing, CRISPR, Plasmid Preparation, Negative Control, Transfection, Comparison, Western Blot

Journal: bioRxiv

Article Title: Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 with functional confirmation

doi: 10.1101/2024.04.02.587857

Figure Lengend Snippet: Representative 10X fluorescent images staining for NTSR2 and Cav2.3 of negative control or CRISPR-Cas9 treated hDRG cultures. A . Peripherin (blue) was used as the neuronal marker, the reporter tag (GFP) was seen only in the CRISPR treated cells, while the target of interest is significantly decreased in the perspective CRISPR treated cells. The merge images of all three channels are shown for each negative control and CRISPR treated cells with the corresponding 20X image. B . Data for the mean fluorescent intensity for the targets of interest Cav2.3 (Red), and NTSR2 (Red) was quantified as means ± SEM of n=10 neurons per group in 3 individual replicates. ****P < 0.0001 by unpaired two-tailed t-test. Scale bars = 100μm

Article Snippet: Antibodies used were as follows: Peripherin (chicken 1:1000; EnCor, AB_2284443) Cav2.3 (Rabbit, Alomone labs, 1:100 AB_2039777; lot ACC006AN0302), NTSR2 (rabbit, 1:200, ThermoFisher BS-12004R), TRPV1 (Rabbit, 1:500 Fisher scientific PA1748) GAPDH (mouse, Cell signaling 97166s) secondary Alexa Fluor goat anti-mouse immunoglobulin G (IgG) 594 (1:5000; Invitrogen, A11032, lot 1985396), and secondary Alexa Fluor goat anti-mouse IgG 488 (1:5000; Invitrogen A-11034, lot 2110499).

Techniques: Staining, Negative Control, CRISPR, Marker, Two Tailed Test