Journal: bioRxiv

Article Title: Focal adhesion kinase regulates early steps of myofibrillogenesis in cardiomyocytes

doi: 10.1101/261248

Figure Lengend Snippet: A) Representative SIM micrographs of hCMs spread for indicated times, visualizing F-actin and endogenous α-actinin-2. Insets: enlarged views of dotted yellow boxes. Dotted white lines: leading edge of the hCM. B) Live dynamics of α-actinin-2 during Hcm spreading. Inset: Time montage of dotted yellow box. 0 min corresponds to 6 hours post plating. Yellow and white arrow denote two separate Z-body stitching events. C) hCM expressing α-actinin-2 tdEos spread for 18 hours (corresponding to 0h in inset). Yellow circle represents ROI used for photo-conversion. Time montage shows unconverted α-actinin-2 (gray) overlaid with converted α-actinin-2 (magenta). Yellow arrows follow converted Z-bodies incorporating into Z-lines. D) Representative SIM micrographs of hCMs spread for 6 or 24 hours visualizing F-actin and endogenous paxillin. Insets show enlarged views of dotted yellow boxes. Dotted white lines show leading edge of the spreading hCM. E) Live dynamics of actin (magenta) and paxillin (green) during hCM spreading. 0 hours corresponds to 6 hours post plating. White arrows denote a maturing FA, and orange arrows show an assembling myofibril. F) Correlation between sum adhesion area and median Z-line length performed for time points indicated. Z-bodies (Y-axis): 6 hours-27877 Z-bodies from 80 cells over 3 experiments; 24 hours-33188 Z-bodies from 88 cells over 4 experiments; 1 week-62663 Z-bodies from 145 cells over 3 experiments. Adhesions (X-axis): n= 35, 38 and 28 cells over 4 independent experiments for 6 hours, 24 hours and 1 week, respectively. Error bars represent standard error of the mean. Scale bars: (A, D)-5 μm; (B, C, E)-10 μm.

Article Snippet: mCherry-Alpha-Actinin 2 (54974), tdEos-Alpha-Actinin 2 (57577), tdEos-Paxillin (57653), tdEos-PTK2 (57659), mEos3.2-Vinculin (66952), mEmerald Lifeact (54148) and mCherry-Paxillin (55114) were purchased from Addgene (Cambridge, MA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Focal adhesion kinase regulates early steps of myofibrillogenesis in cardiomyocytes

doi: 10.1101/261248

Figure Lengend Snippet: A) hCM expressing a-actinin-2 mCherry at 27 hours post plating. B) Time montages of region correponding to black arrow displayed at 150 min intervals. Note the appearance of centripetal nucleation from long FA (black arrows). C) Higher temporal sampling shows several examples of the appearance of Z-bodies (orange, black, yellow and white arrows) far away from the initial FA (magenta arrows). Subsequent time frames show the myofibril arising by stitching of Z-bodies (white and black arrows). The adhesions (magenta arrows) subsequently grow brighter as the myofibril incorporates more Z-bodies. Note how Z-body in time frame 0 min (white arrow) is already elongated before the Z-body to the right (yellow arrow) appearing without any observeable from the adhesion at the edge. Scale bar: 10 µm.

Article Snippet: mCherry-Alpha-Actinin 2 (54974), tdEos-Alpha-Actinin 2 (57577), tdEos-Paxillin (57653), tdEos-PTK2 (57659), mEos3.2-Vinculin (66952), mEmerald Lifeact (54148) and mCherry-Paxillin (55114) were purchased from Addgene (Cambridge, MA).

Techniques: Expressing, Sampling

Journal: bioRxiv

Article Title: Focal adhesion kinase regulates early steps of myofibrillogenesis in cardiomyocytes

doi: 10.1101/261248

Figure Lengend Snippet: For measurement of a-actinin-2 Z-line lengths, widefield fluorescence image stacks (gray) were deconvolved and segmented using Nikon Elements Software (colorized binary file). The long axis lengths of Z-bodies were exported to MATLAB and data was filtered and processed to generate median Z-line measurements. For paxillin quantification, SIM micrographs were used for thresholding in FIJI. Filtering and further analysis was performed in MATLAB. Scale bar: 5 µm.

Article Snippet: mCherry-Alpha-Actinin 2 (54974), tdEos-Alpha-Actinin 2 (57577), tdEos-Paxillin (57653), tdEos-PTK2 (57659), mEos3.2-Vinculin (66952), mEmerald Lifeact (54148) and mCherry-Paxillin (55114) were purchased from Addgene (Cambridge, MA).

Techniques: Fluorescence, Software

Journal: bioRxiv

Article Title: Focal adhesion kinase regulates early steps of myofibrillogenesis in cardiomyocytes

doi: 10.1101/261248

Figure Lengend Snippet: A) Validation of FAK knockdown using western blotting and immunofluorescence. For western blotting, tubulin was used as loading control for normalization. N=3 independent knockdowns. Representative SIM micrographs of scrambled control (si-Scr) and FAK depleted (si-FAK) hCMs spread for 24 hours. Endogenous paxillin and phospho-FAK (Y397) staining shows loss of phosphorylated FAK from adhesions and decrease in sum adhesion area. Inset: enlarged view of dotted yellow box. B) F-actin and endogenous α-actinin-2 staining at 24 hours shows overall reduction in Z-line length. Insets: enlarged view of dotted yellow box. Adhesions: sum paxillin area was quantified, as before. n=34 si-Scr and 29 si-FAK cells over 3 experiments. Z-lines: For si-Scr-19196 Z-bodies measured from 107 cells over 4 experiments. For si-FAK-28665 Z-bodies measured from 106 cells over 4 experiments. C) - Representative SIM micrographs of control versus PF-228 treated hCMs spread for 6 hours. Endogenous paxillin and F-actin staining shows increased adhesion area. Endogenous α-actinin-2 and F-actin staining shows longer Z-lines. Adhesion quantification: n=46 PF-228 treated cells over 4 experiments compared to control data set from . Z-line quantification: PF-228-21180 Z-bodies measured from 88 cells over 3 independent experiments, compared to control Z-line data set from . Error bars represent standard error of the means. Scale bars: 5 μm.

Article Snippet: mCherry-Alpha-Actinin 2 (54974), tdEos-Alpha-Actinin 2 (57577), tdEos-Paxillin (57653), tdEos-PTK2 (57659), mEos3.2-Vinculin (66952), mEmerald Lifeact (54148) and mCherry-Paxillin (55114) were purchased from Addgene (Cambridge, MA).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Focal adhesion kinase regulates early steps of myofibrillogenesis in cardiomyocytes

doi: 10.1101/261248

Figure Lengend Snippet: A) Half-lives and mobile fractions of FAK, vinculin and paxillin measured using photo-conversion in untreated U2-OS cells, untreated hCMs and PF-228 treated hCMs. FAK: n=15 U2-OS, 14 control hCM and 13 PF-228 treated hCM over 3 independent experiments. Vinculin: n=15 U2-OS, 14 control hCM and 15 PF-228 treated hCM over 3 independent experiments. B) Representative hCM expressing α-actinin-2 mCherry (inverted gray). Kymographs were generated using dotted black line to measure the rate of retrograde flow of Z-bodies. Representative kymographs for control versus PF-228 treated hCMs are depicted. E) Quantification of Z-body flow rates in control versus PF-228 hCMs. Control: 6 cells with 60 trajectories over 3 independent experiments. PF-228: 6 cells with 50 trajectories over 3 independent experiments. Error bars represent standard error of the means. Scale bars: 10 μm.

Article Snippet: mCherry-Alpha-Actinin 2 (54974), tdEos-Alpha-Actinin 2 (57577), tdEos-Paxillin (57653), tdEos-PTK2 (57659), mEos3.2-Vinculin (66952), mEmerald Lifeact (54148) and mCherry-Paxillin (55114) were purchased from Addgene (Cambridge, MA).

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: Focal adhesion kinase regulates early steps of myofibrillogenesis in cardiomyocytes

doi: 10.1101/261248

Figure Lengend Snippet: A) Representative SIM micrographs of hCMs allowed to spread for 6 hours showing endogenous titin and F-actin in control versus PF-228 treatment. Inset: Enlarged view of dotted yellow box. B) Measurement of distance from the edge for the appearance of first titin “rings” around Z-bodies. n=23 cells each for control and PF-228 over 3 experiments. C) Representative SIM micrographs of hCMs allowed to spread for 6 hours showing endogenous ß-myosin II and titin in control versus PF-228 treatment. Inset: Enlarged view of dotted yellow box and representative line scans. Note sarcomeric organization in PF-228 treated hCMs. White boxes were used to generate an averaged line scan for ß-myosin II in control (n=11 cells, 3 experiments) and PF-228 (n=10 cells, 3 experiments) show increased ß-myosin II incorporation into myofibrils upon PF-228 treatment. D) Representative SIM micrographs of hCMs allowed to spread for 6 hours showing endogenous myomesin and titin in control versus PF-228 treatment. Insets: Enlarged view of dotted yellow box and representative line scans. Note sarcomeric organization in PF-228 treated hCMs. E) Representative hCM at 16 hours expressing α-actinin-2 tdEos. Yellow circle represents photo-converted (show in magenta) area. Bar graphs compare half-life and mobile fraction upon PF-228 treatment. Error bars represent standard error of the mean. Scale bars: (A, C, D)-5 μm; (E)-10 μm.

Article Snippet: mCherry-Alpha-Actinin 2 (54974), tdEos-Alpha-Actinin 2 (57577), tdEos-Paxillin (57653), tdEos-PTK2 (57659), mEos3.2-Vinculin (66952), mEmerald Lifeact (54148) and mCherry-Paxillin (55114) were purchased from Addgene (Cambridge, MA).

Techniques: Expressing

Journal: Ecotoxicology and environmental safety

Article Title: Additive cardiotoxicity of a bisphenol mixture in zebrafish embryos: The involvement of calcium channel and pump.

doi: 10.1016/j.ecoenv.2023.115225

Figure Lengend Snippet: Fig. 4. Effects of BPs on cardiomyocyte development. Representative confocal microscopy image of TG(myl7:NLS-mCherry) ZFEs at 48 hpf exposed to 0.05% DMSO (A) or Mix 2 (BPA 8.7 µM + BPF 18 µM + BPAF 1.3 µM + BPB 4.2 µM), which induced approximately 40% heart rate reduction (B). BP exposure did not affect the cardiomyocyte count (C), based on the t test (n = 10). BP exposure, which induced approximately 30% heart rate reduction, did not affect mRNA expression of genes related to cardiomyocyte development: hand2 (D) and nkx20.5 (E). Only BPF and BPAF significantly downregulated bmp4 mRNA expression (F), based on the one- way ANOVA test followed by Dunnett’s post hoc analysis (n = 3). SC: DMSO 0.05%, BPA: 25 µM, BPF: 60 µM, BPAF: 4 µM, BPB: 12 µM, Mix 1: BPA 6.6 µM + BPF 13.6 µM + BPAF 1 µM + BPB 3.2 µM. ns, P ≥0.03; *, P ≤0.03; **, P ≤0.002; *** , P ≤0.0002; *** *, P ≤0.0001. The data are presented as means ± standard deviations.

Article Snippet: Embryos were stained with mCherry Mouse Polyclonal Antibody (Cusabio, Houston, TX, USA), Goat Anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, and Alexa Fluor Plus 594 (Invitrogen) according to a previously described protocol (Hammond-Weinberger and ZeRuth, 2020).

Techniques: Confocal Microscopy, Expressing