Journal: iScience

Article Title: α- and β-myosin II can be non-uniformly distributed within the cardiac sarcomere

doi: 10.1016/j.isci.2025.112233

Figure Lengend Snippet: α- and β-myosin II can exhibit unique distributions within cardiac sarcomeres (A) Representative example of IF of endogenous β-myosin II (top) and expression of mEGFP- MYH6 (middle) in a hiCM; bottom shows overlay. Scale bars: left 10 μm, right 2 μm. (B) Line scan from yellow boxes in (A). (C) Average of all line scans (20 cells, 772 sarcomeres, 3 independent experiments). Lines show population-averaged mean value while shaded bands show ± standard error of the mean. (D) Top: expression of mNeon- MYH7 and IF of endogenous α-myosin II in a representative hiCM. Bottom: Average of all line scans (15 cells, 456 sarcomeres, 3 independent experiments). Lines show population-averaged mean value while shaded bands show ± standard error of the mean. White brackets denote individual filament stacks. Scale bar: 2 μm. (E) Distance each myosin II extended from filament center in hiCMs (β-myosin II IF: 14 cells, 280 measurements; α-myosin II IF: 15 cells, 300 measurements; mNeon- MYH7 (β expressed): 15 cells, 300 measurements; mEGFP- MYH6 (α expressed): 15 cells, 300 measurements). p -values in grey (Welch's t-test). (F) Simplified model of the potential arrangement of β-myosin II (magenta) and α-myosin II (green) in thick filaments of iPSC-derived cardiac myocyte sarcomeres. (G) Left: IF using antibodies against β-myosin II (top) and α-myosin II (bottom) in different sections of human ventricle; right: distance the fluorescent signal of each myosin extended from the filament center (β-myosin II: 280 measurements; α-myosin II: 321 measurements). Scale bar: 2 μm. p -values in grey (Welch's t-test). (H) Left: individual β-myosin II (ventricle) and α-myosin II (atrium) whole-mount immunofluorescence stains in 72 h post-fertilization zebrafish embryos; right: distance the fluorescent signal of each myosin extended from the filament center (β-myosin II: 14 embryos, 222 measurements; α-myosin II: 13 embryos, 102 measurements). White brackets in each image denote individual filament stacks. Scale bar: 2 μm. p -values in grey (Welch's t-test); (I) AlphaFold3-predicted model of α-myosin II/β-myosin II heavy chain ( MYH6 / MYH7 genes, respectively) motors + motor/lever arms, modeled with MYL2 / MYL3 (gray), which encode the ventricular essential/regulatory myosin light chains (a similar AlphaFold3-predicted model, but modeled with atrial essential/regulatory myosin light chains instead of ventricular light chains, is shown in ). N1, 2, and 3 in (E), (G), and (H) represent individual biological replicates, color-coded by replicate.

Article Snippet: In brief, the hiCMs were thawed and plated in 100uL per well Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics; catalog #R1151) at a density of 50,000 cells per well in 96-well polystyrene cell-culture plates pre-coated for 2 h with gelatin (EMD Millipore; catalog # ES-006-B).

Techniques: Expressing, Derivative Assay, Immunofluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Mycobacterium tuberculosis PE31 ( Rv3477 ) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1

doi: 10.3389/fcimb.2020.00040

Figure Lengend Snippet: Ms_PE31 can alter the surface characteristics of M. smegmatis . (A) PE31 (Rv3477) sequence amplified by PCR, applying M. tuberculosis genome, ~297 bp in length (lane 1 = DNA ladder and lane 2 = amplified gene). (B) The Ace-induced recombinant cell lysates were prepared and employed the Western blot for confirmation of His-tagged PE31 protein expression. (C) Both recombinant strains cultured on plates containing 7H9 solid media with hyg and Ace (0.02%, w/v), the developed single colony (D) Recombinant strains cultured in Ace (0.02%, w/v) added 7H9 liquid on polystyrene plates to induce the development of biofilm. (E) The quantification of biofilm formation was confirmed by Tetrahydrofuran (THF) assay ( n = 3). Results were determined by Student's t -test, * P < 0.05. Error bars represent the standard deviation of mean.

Article Snippet: Then, took 100 μl at mentioned time points and dappled onto Middlebrook 7H9 solid media plates containing Hyg by 10-fold serial dilution.

Techniques: Sequencing, Amplification, Recombinant, Western Blot, Expressing, Cell Culture, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Mycobacterium tuberculosis PE31 ( Rv3477 ) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1

doi: 10.3389/fcimb.2020.00040

Figure Lengend Snippet: Ms_PE31 increase growth under multiple stress. (A) The growth rate of recombinant strains was measured under in-vitro low pH condition. Cultured bacteria were harvested and re-cultured in 7H9 (pH = 5) to maintained the OD 600 =0.8. Then, incubated in 37°C and 100 μl taken from it after different time intervals for workable enumeration ( n = 3). (B) The reactive nitrogen stress was examined by spot test, the supplementation of 2 and 5 mM diamide to the 7H9 solid medium to grown the Ms_Vec and Ms_PE31 strains. Growth of bacteria on 7H9 solid media which contained mentioned concentrations of diamide ( n = 3). (C,D) Ms_Ves and Ms_PE31 survival upon exposure to H2O2 and SDS, respectively, examined by disk diffusion technique. Whatman disks used to mottled the different concentrations of H2O2 (10 μl) and SDS (10 μl) ( n = 3). Area of the zone of inhibition was calculated after 3−4 days incubation at 37°C. The results were determined by Student's t -test, * P < 0.05 and ** P < 0.01. Error bars represent the standard deviation of mean.

Article Snippet: Then, took 100 μl at mentioned time points and dappled onto Middlebrook 7H9 solid media plates containing Hyg by 10-fold serial dilution.

Techniques: Recombinant, In Vitro, Cell Culture, Bacteria, Incubation, Spot Test, Diffusion-based Assay, Inhibition, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Mycobacterium tuberculosis PE31 ( Rv3477 ) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1

doi: 10.3389/fcimb.2020.00040

Figure Lengend Snippet: Survival rate of M. smegmatis recombinant strains. (A) Recombinant strains infected macrophages were laved and lysed SDS (0.025%, w/v) at indicated intervals. 10-fold serial diluted lysed cells were mottled on hyg containing 7H9 solid medium plates. After 3–4 days, CFU was computed ( n = 3). (B) Growth kinetics of recombinant strains were determined by the growth of bacteria in 7H9 liquid added Ace (1%, w/v), Tw (0.05%, v/v) and hyg (100 μg/ml) ( n = 3). Results were determined by Student's t -test, *** P < 0.001. Error bars represent the standard deviation of mean.

Article Snippet: Then, took 100 μl at mentioned time points and dappled onto Middlebrook 7H9 solid media plates containing Hyg by 10-fold serial dilution.

Techniques: Recombinant, Infection, Bacteria, Standard Deviation