Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MYLK4 promotes tumor progression through the activation of epidermal growth factor receptor signaling in osteosarcoma

doi: 10.1186/s13046-021-01965-z

Figure Lengend Snippet: MYLK4 promotes osteosarcoma cell metastasis in vivo and in vitro. a The MYLK4 overexpression effects were confirmed by western blotting. b The effect of MYLK4 overexpression on OS cell migration was assessed by a wound healing assay. Representative images of migration were shown in the left panel. The degrees to which the wounds healed in the indicated groups were shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01, Scale bars = 250 μm. c The effect of MYLK4 overexpression on OS cell invasion was assessed by a transwell assay (left panel). The relative proportions of invading cells were shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01, Scale bars = 200 μm. d The MYLK4 knockdown effects were confirmed by western blotting. e The effect of MYLK4 knockdown on OS cell migration was assessed by a wound healing assay. Representative images of migration were shown in the left panel. The degrees to which the wounds healed in the indicated groups were shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01, Scale bars = 250 μm. f The effect of MYLK4 knockdown on OS cell invasion was assessed by a transwell assay (left panel). The relative proportions of invading cells were shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01, Scale bars = 200 μm. g The expression of EMT markers was detected by western blotting in MYLK4 over-expression and MYLK4-knockdown OS cells. h The number of lung metastasis nodules formed by MYLK4-knockdown 143B cells and control cells in orthotopic osteosarcoma implanted mice was shown in the right panel. Statistically significant differences (t-test), ** P < 0.01. Representative images of lung morphology and lung metastatic nodules formed by MYLK4-knockdown 143B cells and control cells were shown in the left panel. Corresponding HE staining was shown in the middle panel. Scale bars = 100 μm

Article Snippet: Primary antibodies: P-EGFR (Tyr1068) (#3777, Cell Signaling), EGFR (#4267, Cell Signaling), P-AKT (#4060, Cell Signaling), AKT (#4685, Cell Signaling), P-ERK (#4370, Cell Signaling), ERK (#4695, Cell Signaling), MYLK4 (24309–1-AP, Proteintech).

Techniques: In Vivo, In Vitro, Over Expression, Western Blot, Migration, Wound Healing Assay, Transwell Assay, Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MYLK4 promotes tumor progression through the activation of epidermal growth factor receptor signaling in osteosarcoma

doi: 10.1186/s13046-021-01965-z

Figure Lengend Snippet: MYLK4 promotes osteosarcoma cell proliferation. a Osteosarcoma tissues from mice with tumor xenografts inoculated with MYLK4-knockdown 143B cells. The weight of osteosarcoma samples was shown in the down panel. Statistically significant differences (t-test), ** P < 0.01. b Western blotting were performed to detect the shRNA interference efficiency in vivo. c The expression of Ki67 was detected by IHC in orthotopic OS implanted-mice tumor tissues formed by MYLK4-knockdown 143B cells and control cells. The results of Ki67 positive (%) were shown in the right panel. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01, Scale bars = 100 μm. d The effect of MYLK4 overexpressing on OS cell proliferation was assessed by the CCK8 assay. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01. e The effect of MYLK4 overexpression on colony formation in OS cells. Representative images of colony formation were shown in the left panel. The numbers of cells in the indicated groups are shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01. f Cell proliferation was measured by CCK8 assay with/without MYLK4 knockdown in OS cells. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01. g The effect of MYLK4 knockdown on colony formation in OS cells. Representative images of colony formation were shown in the left panel. The numbers of cells in the indicated groups were shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01

Article Snippet: Primary antibodies: P-EGFR (Tyr1068) (#3777, Cell Signaling), EGFR (#4267, Cell Signaling), P-AKT (#4060, Cell Signaling), AKT (#4685, Cell Signaling), P-ERK (#4370, Cell Signaling), ERK (#4695, Cell Signaling), MYLK4 (24309–1-AP, Proteintech).

Techniques: Western Blot, shRNA, In Vivo, Expressing, CCK-8 Assay, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MYLK4 promotes tumor progression through the activation of epidermal growth factor receptor signaling in osteosarcoma

doi: 10.1186/s13046-021-01965-z

Figure Lengend Snippet: MYLK4 interacts with EGFR. a Silver staining of whole cell lysates (WCL) and immunoprecipitates (IP) of FLAG-MYLK4 143B cells. The indicated bands were sequenced by mass spectrometry (MS) analysis. b Secondary peptide structure of EGFR acquired by MS. c 143B cells were transfected with Flag-tagged MYLK4 construct and the interaction between exogenous MYLK4 and EGFR was detected by IP and immunoblotting (IB). d Interaction between endogenous MYLK4 and EGFR by IP analyses in 143B cells. e HEK-293 T cells were cotransfected with Flag-tagged MYLK4 and HIS-tagged EGFR constructs, and the interaction between exogenous MYLK4 and EGFR was detected by IP and IB

Article Snippet: Primary antibodies: P-EGFR (Tyr1068) (#3777, Cell Signaling), EGFR (#4267, Cell Signaling), P-AKT (#4060, Cell Signaling), AKT (#4685, Cell Signaling), P-ERK (#4370, Cell Signaling), ERK (#4695, Cell Signaling), MYLK4 (24309–1-AP, Proteintech).

Techniques: Silver Staining, Mass Spectrometry, Transfection, Construct, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MYLK4 promotes tumor progression through the activation of epidermal growth factor receptor signaling in osteosarcoma

doi: 10.1186/s13046-021-01965-z

Figure Lengend Snippet: MYLK4 promotes EGFR phosphorylation and the downstream signaling pathway. a The expression of p-EGFR, p-AKT and p-ERK was detected by western blotting in MYLK4-overexpressing cells. b The expression of p-EGFR, p-AKT and p-ERK was detected by western blotting in MYLK4-knockdown cells. c The expressions of p-EGFR, p-AKT and p-ERK were detected by western blotting with/without EGF stimulation for 15 min. d The expression of p-EGFR was detected by western blotting with EGF (50 ng/ml) stimulation for different time period respectively in MYLK4-knockdown and the control cells. e The expressions of MYLK4 and P-EGFR (Y1068) expression in human OS tissues were detected by IHC assay. Scale bars = 200 μm. f The score of MYLK4 and P-EGFR (Y1068)-positive staining were quantified and the Spearman correlation was performed

Article Snippet: Primary antibodies: P-EGFR (Tyr1068) (#3777, Cell Signaling), EGFR (#4267, Cell Signaling), P-AKT (#4060, Cell Signaling), AKT (#4685, Cell Signaling), P-ERK (#4370, Cell Signaling), ERK (#4695, Cell Signaling), MYLK4 (24309–1-AP, Proteintech).

Techniques: Expressing, Western Blot, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MYLK4 promotes tumor progression through the activation of epidermal growth factor receptor signaling in osteosarcoma

doi: 10.1186/s13046-021-01965-z

Figure Lengend Snippet: The inhibitor of MYLK4 shows synergistic effect with the EGFR inhibitor in OS in vitro and in vivo. a 143B and b HOS cells treated with Gefitinib alone, ML-7 alone, or Gefitinib/ML-7 in combination. Cell viability was determined by CCK-8 assay. Combination index (CI) was calculated by using CompuSyn software. c Divide cells into four groups, Control group, Gefitinib alone group, ML-7 alone group and Gefitinib/ML-7 in combination group. Colony-formation assay was carried out in those four groups. Representative images of colony formation are shown in the upper panel. The numbers of cells in the indicated groups were shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01. d Wound healing assay was carried out in those four groups. Representative images of migration were shown in the upper panel. The degrees to which the wounds healed was shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01, Scale bars = 250 μm. e Transwell assay was carried out in those four groups. Representative images of invasion were shown in the upper panel. The proportions of invading cells were shown in the histogram. The bars indicate the mean ± s.d. Statistically significant differences (t-test), ** P < 0.01, Scale bars = 200 μm. f The number of lung metastasis nodules formed by 143B cells in orthotopic osteosarcoma implanted mice was shown in the right panel, the mice were divided into four groups: mice treated with dmso only, mice treated with Gefitinib only, mice treated with ML-7 only and mice treated with Gefitinib plus ML-7. Statistically significant differences (t-test), ** P < 0.01. Representative images of lung morphology and lung metastatic nodules in the four groups were shown in the left panel. Scale bars = 250 μm. g Tumor weights of the tumors in the four groups were recorded. Statistically significant differences (t-test), ** P < 0.05. h The expressions of p-EGFR, p-AKT and p-ERK in vitro were measured by western blotting in different groups. i The expressions of p-EGFR, p-AKT and p-ERK in tumor xenograft tissues were measured by western blotting

Article Snippet: Primary antibodies: P-EGFR (Tyr1068) (#3777, Cell Signaling), EGFR (#4267, Cell Signaling), P-AKT (#4060, Cell Signaling), AKT (#4685, Cell Signaling), P-ERK (#4370, Cell Signaling), ERK (#4695, Cell Signaling), MYLK4 (24309–1-AP, Proteintech).

Techniques: In Vitro, In Vivo, CCK-8 Assay, Software, Colony Assay, Wound Healing Assay, Migration, Transwell Assay, Western Blot

Journal: Stem cell research & therapy

Article Title: JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes.

doi: 10.1186/s13287-023-03610-2

Figure Lengend Snippet: Fig. 1 JAK2 and CD200 as potential surface markers for ventricular-specific cardiomyocytes. A Schematic diagram illustrating the protocol used to identify putative surface markers for ventricular-specific CMs. Created with BioRender.com. B Gating strategy for isolation of MLC2A + and MLC2V + cardiomyocytes derived from BJ and ESO3 lines by FACS. C Hierarchical clustering analysis of the top 500 differentially expressed genes revealed a distinct separation between MLC2V + and MLC2A + samples. D Heatmap showing the list of putative surface marker genes for ventricular-specific cardiomyocytes identified through RNA sequencing analysis

Article Snippet: The following primary antibodies were used: rabbit-antihuman MLC2V (1:1000; 10,906–1-AP; Proteintech), mouse-anti-human MLC2A (1:1000; 311 011; Sypnatic Systems), rabbit-anti-GAPDH (1:1000; D16H11, Cell Signalling Technologies) and mouse-anti-GAPDH (1:1000; ab16048, Abcam).

Techniques: Isolation, Derivative Assay, Marker, RNA Sequencing

Journal: Stem cell research & therapy

Article Title: JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes.

doi: 10.1186/s13287-023-03610-2

Figure Lengend Snippet: Fig. 3 SIRPA+/CD200 + population shows enrichment of ventricular CMs. A Representative FACS plots of CMs sorted using SIRPA and CD200 antibodies. Unstained CMs served as negative control. B Representative images of SIRPA+/CD200 + (top panel) and SIRPA+/CD200− (bottom panel) CMs co-stained with MLC2V and MLC2A antibodies. Nucleus were stained with DAPI The percentage of MLC2V + and MLC2A + CMs in the respective populations was quantified and expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (t test). Data were collected from duplicate experiments. Scale bar = 100 µm. C Western blot analysis of MLC2A and MLC2V expression in SIRPA+/CD200 + and SIRPA+/CD200− populations, normalized to GAPDH expression. Full-length blots are presented in Additional file 6: Fig. S3 (for BJ SIRPA/CD200 sorted samples) and Additional file 7: Fig. S4 (for H7 SIRPA/CD200 sorted samples)

Article Snippet: The following primary antibodies were used: rabbit-antihuman MLC2V (1:1000; 10,906–1-AP; Proteintech), mouse-anti-human MLC2A (1:1000; 311 011; Sypnatic Systems), rabbit-anti-GAPDH (1:1000; D16H11, Cell Signalling Technologies) and mouse-anti-GAPDH (1:1000; ab16048, Abcam).

Techniques: Negative Control, Staining, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Generation of Highly Purified Human Cardiomyocytes from Peripheral Blood Mononuclear Cell-Derived Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0126596

Figure Lengend Snippet: A: Schematic diagram demonstrating the main steps of the procedure used for the direct differentiation of iPS cells to cardiomyocytes. The main components of the media applied are noted above the time line. Cell suspensions were kept on an orbital shaker at 5% O2 and 7% CO2 from day 1 to 14. Then the flasks with the cell aggregates were moved to an orbital shaker at 20% O2 and 7% CO2; B: Immunofluorescent staining of cardiomyocytes with α-actinin (ActN2), cardiac troponin T (cTNT), myosin light chain 2 (MLC2v), and N cadherin (NCad)(scale bar 25 μm), cardiac troponin T and connexin 43 (Con43)(scale bar 50 μm); C: TNNT2 and MYL2 gene expression in iPS cell-derived cardiomyocytes. Gene expression for both genes increases until day 25 of differentiation.

Article Snippet: After fixation, the cells were washed with Permwash (BD Biosciences) and stained with primary antibody dilutions for rabbit anti-cardiac troponin T (Abcam, 1:200) mouse anti-α-actinin (Abcam, 1:1000), rabbit anti-myosin light chain 2 (MLC2v) (Proteintech, 1:200), rabbit anti-connexin 43 (Abcam, 1:1000) and rabbit anti-N cadherin (Abcam, 1:200) overnight.

Techniques: Staining, Gene Expression, Derivative Assay

Journal: Stem Cells International

Article Title: Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers

doi: 10.1155/2016/2634013

Figure Lengend Snippet: IMR hiPS-cardiomyocytes (CMs) cultured on aligned fiber (AF)-coated and gelatin-coated (Flat) microelectrode arrays (MEA). (a) Phase contrast images of CMs plated on the MEA for 2 days: the scale bar in = 200 μ m. (b, c) Fluorescence images of CMs cultured for 14 days on MEA. (b) MLC2v is shown in red, (c) β -MHC in green, and DAPI in blue. The scale bar = 200 μ m. The color on fiber is due to autofluorescence of PMGI. White arrow marked the fiber orientation.

Article Snippet: CMs derived from IMR hiPSCs were fixed with 4% paraformaldehyde and were permeabilized in PBS plus 0.5% Triton X-100 for 0.5 h. After blocking in a 5% normal goat serum, 5% normal donkey serum, 3% BSA, and 0.1% Tween 20 in PBS for 16 h at 4°C, the cells were incubated with an α -actinin (mouse monoclonal IgG, 1 : 1000; Sigma), troponin T2 (TnT2, mouse monoclonal IgG, 1 : 200, Santa Cruz Biotechnology, Dallas, TX, USA), MLC2v (rabbit polyclonal IgG, 1 : 200; Proteintech Group, Rosemont, IL, USA), or β -MHC (mouse MYH7 monoclonal IgM, 1 : 100; Santa Cruz Biotechnology) antibody for 16 h at 4°C.

Techniques: Cell Culture, Fluorescence

Journal: Nature methods

Article Title: Chemically Defined and Small Molecule-Based Generation of Human Cardiomyocytes

doi: 10.1038/nmeth.2999

Figure Lengend Snippet: Characterization of atrial vs. ventricular profile of cardiomyocytes produced under chemically defined conditions. a) Flow cytometry assessment of expression of cardiac troponin T (TNNT2), atrial myosin light chain 2 (MLC2A), and ventricular myosin light chain (MLC2V) in cardiomyocytes derived and maintained in CDM3 at differentiation from day 10 through day 60, n = 3. Error bars represent S.E.M. Additional data is provided in . b ) Immunofluorescence staining of day 20 cardiomyocytes with the same antibodies used for flow cytometry to demonstrate specificity. Scale bar, 12.5 µm.

Article Snippet: Cells were transferred to flow cytometry tubes (BD Biosciences) and fixed with 1% PFA for 15 min, permeabilized with 90% methanol for 15 min, and stained using 1:200 rabbit polyclonal IgG POU5F1 (H-134, sc-9081, Santa Cruz Biotechnology), 1:20 mouse monoclonal IgG3 SSEA4-APC (clone MC-813-70, FAB1435A, R&D Systems), 1:200 NANOG rabbit polyclonal IgG (H-155, sc-33759, Santa Cruz Biotechnology), 1:20 mouse monoclonal IgM TRA-1–60-FITC (FAB4770P, R&D Systems), 1:200 mouse monoclonal IgG1 TNNT2 (cardiac troponin T, 13-11, MS-295-P, Thermo Scientific), 1:200 mouse monoclonal IgG1 TNNT2 (cardiac troponin T, clone 1C11, ab8295, Abcam), 1:200 mouse monoclonal IgG2b TNNI (cardiac and skeletal troponin I, clone 2Q1100, T8665-16M, US Biological), 1:400 mouse monoclonal IgG2b MLC2A (MYL7, Synaptic Systems), and 1:200 rabbit polyclonal IgG MLC2V (MYL2, ProteinTech) for 45 min at RT.

Techniques: Produced, Flow Cytometry, Expressing, Derivative Assay, Immunofluorescence, Staining