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: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Berberine promotes lacteal junction zippering and ameliorates diet-induced obesity through the RhoA/ROCK signaling pathway.

doi: 10.1016/j.phymed.2023.155268

Figure Lengend Snippet: Fig. 4. Berberine inhibits intestinal RhoA/ROCK1 signaling in DIO mice. (A) Representative Western Blot images of relative proteins in RhoA/ROCK1 signaling pathway. (B-D) Expression levels of P-RhoA/RhoA, ROCK1, P-MLC2/MLC2 in Western Blot (n = 6/group). (E-F) Representative IHC images of ROCK1 and the corresponding mean optical density. Scale bars: 100 μm and 25 μm (n = 4/group). (G-H) Representative IF images of P-MLC2 and the corresponding mean fluo rescence intensity. Scale bar: 100 μm (n = 4/group). (I-J) Representative TUNEL images of the small intestine and the corresponding apoptotic index. Scale bars: 50 μm and 25 μm (n = 4/group). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the NC group; ###p < 0.001 compared with the DIO group.

Article Snippet: The primary antibodies included P-RhoA (AF8020, Affinity, 1:1000), RhoA (2117, CST, 1:1000), ROCK1 (4035, CST, 1:1000), MLC2 (8505, CST, 1:1000), P-MLC2 (3675, CST, 1:1000), Ephrin B2 (A11349, ABclonal, 1:1000), ephrin receptor B4 (EPHB4, A3293, ABclonal, 1:1000), vascular endothelial growth factor C (VEGFC, 22601-1-AP, Proteintech, 1:1000), vascular endothelial growth factor receptor- 2 (VEGFR2, 26415-1-AP, Proteintech, 1:1000), VEGFR3 (20712-1-AP, Proteintech, 1:1000), delta-like ligand 4 (DLL4, 21584-1-AP, Proteintech, 1:1000), forkhead box C1 (FOXC1, 55365-1-AP, Proteintech, 1:1000), FOXC2 (23066-1-AP, Proteintech, 1:1000), Vinculin (66305-1- Ig, Proteintech, 1:1000).

Techniques: Western Blot, Expressing, TUNEL Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Berberine promotes lacteal junction zippering and ameliorates diet-induced obesity through the RhoA/ROCK signaling pathway.

doi: 10.1016/j.phymed.2023.155268

Figure Lengend Snippet: Fig. 7. Berberine inhibits the expression of RhoA/ROCK1 signaling pathway in HDLECs. (A) Representative Western Blot images of relative proteins in RhoA/ROCK1 signaling pathway in HDLEC. (B-D) Expression levels of P-RhoA/RhoA, ROCK1, P-MLC2/MLC2 in western blot. (n = 3/group). (E-H) IF staining of ROCK1 and P- MLC2, and the respective mean fluorescence intensity. Scale bar: 100 μm (n = 3/group). (I-J) IF staining of vinculin and the corresponding mean fluorescence intensity. Scale bar: 50 μm (n = 3/group). (K-L) Representative Western Blot images of vinculin and statistical analysis of protein expression level (n = 3/group). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the Con group.

Article Snippet: The primary antibodies included P-RhoA (AF8020, Affinity, 1:1000), RhoA (2117, CST, 1:1000), ROCK1 (4035, CST, 1:1000), MLC2 (8505, CST, 1:1000), P-MLC2 (3675, CST, 1:1000), Ephrin B2 (A11349, ABclonal, 1:1000), ephrin receptor B4 (EPHB4, A3293, ABclonal, 1:1000), vascular endothelial growth factor C (VEGFC, 22601-1-AP, Proteintech, 1:1000), vascular endothelial growth factor receptor- 2 (VEGFR2, 26415-1-AP, Proteintech, 1:1000), VEGFR3 (20712-1-AP, Proteintech, 1:1000), delta-like ligand 4 (DLL4, 21584-1-AP, Proteintech, 1:1000), forkhead box C1 (FOXC1, 55365-1-AP, Proteintech, 1:1000), FOXC2 (23066-1-AP, Proteintech, 1:1000), Vinculin (66305-1- Ig, Proteintech, 1:1000).

Techniques: Expressing, Western Blot, Staining, Fluorescence

Journal: Molecular Cancer

Article Title: RHOA lactylation at oncogenic hotspots promotes oncogenic activity and protein stabilization

doi: 10.1186/s12943-025-02511-7

Figure Lengend Snippet: K118 lactylation contributes to accumulation of the active GTP-bound RHOA. a Alignment of the lactylation site and adjacent protein sequences of RHOA in different species. b , c ROCK2-RBD-HA was co-expressed with WT (WT), lactylated ( b ), or different mutant RHOA ( c ) in HEK293T cells, and the immunoprecipitated ROCK2-RBD-HA was examined by Western blotting. d WT, lactylated, and mutated RHOA proteins were loaded with GTP, and the rate of intrinsic GTP hydrolysis was analyzed by continuous measurement of phosphate using a colorimetric assay. The concentration of released phosphate was plotted versus time. e – g Expression of p-MLC2 and MLC2 in HEK293T with WT-RHOA, lactylated RHOA ( e ), K118Q ( f ), or K162Q mutant ( g ) was examined by Western blotting. h , i Expression of p-MLC2 and MLC2 in shRHOA-expressing MDA-MB231 ( h ) and SUM159 cells ( i ) with WT, lactylated or different mutated RHOA expression was examined by Western blotting. j F-actin was stained with Rhodamine Phalloidin (Red) in shRHOA-expressing MDA-MB231 cells with stable WT-RHOA or K118Q mutant expression. k Interactions of K118 within the G4 motif and K162 within the G5 motif with GDP-bound RHOA (PDB: 1FTN). Hydrogen bond interactions are shown as dashed lines. l Overlay of WT-RHOA (tan), K118Q (sky blue), K118R (pink), and K118N mutants (light green) after modeling. A magnified overlay of GTP structure was shown on the right. m , n Overlay ( m ) or histogram ( n ) for protein backbone Cα RMSF plots of MD trajectories in WT-RHOA, K118Q, K118R, and K118N mutants. o Heatmap for hydrogen-bonding interactions of GTP with WT-RHOA, K118Q, K118R, and K118N mutants. Occupancy is defined as the ratio of the simulation time that a particular hydrogen bond is present

Article Snippet: The DNA fragments of Mb-Pyl Klacr-RS/Pyl-tRNA pair were synthesized by Beijing Tsingke Biotech Co., Ltd. Antibodies against RHOA and MLC2 were purchased from Proteintech (catalog no. 10749–1-AP and 10,906–1-AP).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Colorimetric Assay, Concentration Assay, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: GYY4137, as a slow-releasing H 2 S donor, ameliorates sodium deoxycholate–induced chronic intestinal barrier injury and gut microbiota dysbiosis

doi: 10.3389/fphar.2024.1476407

Figure Lengend Snippet: The effects of SDC and GYY4137 on TJ expression and localization and the status of MLCK-P-MLC2 signalling pathway in Caco-2 monolayers. Caco-2 monolayers were pretreated with 200 μ M GYY4137 for 48 h and then treated with 200 μ M GYY4137 in the presence or absence of 1.0 mM SDC for 32 h. The total protein of monolayers was collected after treatment for Western blot. (A) GYY4137 or exposure of 1.0 mM SDC for 32 h had no significant effect on the expression level of TJ proteins. (B) Occludin and ZO-1 were stained by immunofluorescence. GYY4137 ameliorated the altered localization of TJs caused by SDC. (C, D) GYY4137 significantly inhibited the increased expression of MLCK and the increased phosphorylation level of MLC2 induced by SDC. N = 3. * P < 0.05 vs. control. # P < 0.05 vs. SDC. ANOVA, Tukey’s test.

Article Snippet: The primary antibodies were purchased from the companies as follows: ZO-1 (source: mouse, Proteintech, United States); Occludin (source: rabbit, CST, United States of America); MLCK (source: rabbit, Proteintech, United Kingdom); MLC2 (source: rabbit, ABclonal, China); and P-MLC2 (Ser19) (source: rabbit, CST, United States).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Phospho-proteomics, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Elevated SPARC Disrupts the Intestinal Barrier Integrity in Crohn's Disease by Interacting with OTUD4 and Activating the MYD88/NF-κB Pathway.

doi: 10.1002/advs.202409419

Figure Lengend Snippet: Figure 3. Repression of SPARC expression improves the integrity of the intestinal epithelial barrier in an inflammatory setting. A) Proteomic analysis of sorted intestinal epithelial cells from DSS colitis mice. B) Representative TEM images of the colon from DSS colitis mice, the arrows represent TJ. Scale bars, 1 μm. C,D): Microvilli length (C) and AJC space (D) of colonic epithelial cells were measured based on the TEM images. E) Representative immunofluorescence images of ZO-1 and occludin in the colon of DSS colitis mice. Scale bars, 50 μm. F) The protein expression of ZO-1 and occludin in the colon of DSS colitis mice was detected by western blotting. G)The concentration of serum FD4. H) Experimental design for a mouse colonic organoid model. I) Representative immunofluorescence images of ZO-1 and occludin in mouse colonic organoids. Scale bars, 50 μm. J) Colonic organoids Intestinal crypt cells were obtained from WT and SPARC-KO mice and organoids were observed daily. On day 7, the organoids were stimulated with

Article Snippet: Primary antibodies specific to SPARC (1:1000, CST, #8725), ZO-1 (1:10000, Proteintech, #21773-1-AP), Occludin(1:10000, Proteintech, #66378-1-Ig), OTUD4 (1:1000, Proteintech, #25070-1-AP), P65 (1:1000, CST, #8242), p-P65 (1:1000, CST, #3033), MLCK(1:1000, AiFang, #AFW8041), MLC2(1:2000, Proteintech, #10906-1- AP), p-MLC2(1:1000, CST, #3674), β-actin(1:20000, Proteintech, #66009- 1-Ig), Lamin B1(1:2000, Proteintech, #12987-1-AP), and GAPDH (1:20000, Proteintech, #60004-1-Ig) were used.

Techniques: Expressing, Western Blot, Concentration Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Elevated SPARC Disrupts the Intestinal Barrier Integrity in Crohn's Disease by Interacting with OTUD4 and Activating the MYD88/NF-κB Pathway.

doi: 10.1002/advs.202409419

Figure Lengend Snippet: Figure 4. SPARC directly combined with OTUD4 A) Schematic of the purification of SPARC-binding proteins in Caco-2 cell using the LC‒MS/MS system. B) Four putative ubiquitin-associated proteins interacted with SPARC were shown. C) Identification of OTUD4 protein by secondary mass spectrometry. D)Co-IP analysis showed that Flag-tagged SPARC interacted with Myc-tagged OTUD4 in Caco-2 cells. E) Immunofluorescence analysis showed colo- calization of SPARC and OTUD4 in Caco-2 cells. Scale bars, 20 μm. F) Immunofluorescence analysis showed colocalization of SPARC and OTUD4 in human colon mucosa tissues. Scale bars, 50 μm. G) OTUD4 and SPARC prediction structures were generated by Alphafold. OTUD4 was represented as

Article Snippet: Primary antibodies specific to SPARC (1:1000, CST, #8725), ZO-1 (1:10000, Proteintech, #21773-1-AP), Occludin(1:10000, Proteintech, #66378-1-Ig), OTUD4 (1:1000, Proteintech, #25070-1-AP), P65 (1:1000, CST, #8242), p-P65 (1:1000, CST, #3033), MLCK(1:1000, AiFang, #AFW8041), MLC2(1:2000, Proteintech, #10906-1- AP), p-MLC2(1:1000, CST, #3674), β-actin(1:20000, Proteintech, #66009- 1-Ig), Lamin B1(1:2000, Proteintech, #12987-1-AP), and GAPDH (1:20000, Proteintech, #60004-1-Ig) were used.

Techniques: Binding Assay, Ubiquitin Proteomics, Mass Spectrometry, Co-Immunoprecipitation Assay, Generated

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Elevated SPARC Disrupts the Intestinal Barrier Integrity in Crohn's Disease by Interacting with OTUD4 and Activating the MYD88/NF-κB Pathway.

doi: 10.1002/advs.202409419

Figure Lengend Snippet: Figure 5. SPARC damages the intestinal epithelial barrier via the MYD88/NF-𝜅B/MLCK pathway A) Ubiquitination assays of MYD88 in cell lysates from HEK293T cells transfected with Flag-SPARC vector and Myc-OTUD4 vector. B) Ubiquitination assays of MYD88 in cell lysates from HEK293T cells transfected with Flag-SPARC vectors with D1 domain deletion mutants and Myc-OTUD4 vector. C-D) Western blotting(C) and immunofluorescence(D) analysis were employed to assess the expression of p65 in the cytoplasm and nucleus of Caco-2 cells following SPARC overexpression or knockdown. Scale bars, 10 μm. E) ChIP analysis of p65 binding to MLCK promoter in Caco-2 cells. Normal mouse IgG was used as a control. F) The MLCK mRNA levels in SPARC overexpression or knockdown Caco-2 cells was detected by RT-qPCR. G) The protein expression of p65, p-p65, MLCK, MLC2, and p-MLC2 in SPARC knockdown or overexpression Caco-2 cells after treatment with TNF-𝛼/IFN-𝛾(T/I) was analyzed by western blot. H) The protein expression of p65, p-p65, MLCK, MLC2, and p-MLC2 in colon tissues from WT or SPARC mice challenged with DSS. I) IHC analysis of SPARC and MLCK protein

Article Snippet: Primary antibodies specific to SPARC (1:1000, CST, #8725), ZO-1 (1:10000, Proteintech, #21773-1-AP), Occludin(1:10000, Proteintech, #66378-1-Ig), OTUD4 (1:1000, Proteintech, #25070-1-AP), P65 (1:1000, CST, #8242), p-P65 (1:1000, CST, #3033), MLCK(1:1000, AiFang, #AFW8041), MLC2(1:2000, Proteintech, #10906-1- AP), p-MLC2(1:1000, CST, #3674), β-actin(1:20000, Proteintech, #66009- 1-Ig), Lamin B1(1:2000, Proteintech, #12987-1-AP), and GAPDH (1:20000, Proteintech, #60004-1-Ig) were used.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Expressing, Over Expression, Knockdown, Binding Assay, Control, Quantitative RT-PCR

Journal: Journal of the American Heart Association

Article Title: Sodium‐Glucose Cotransporter 2 Inhibitor Canagliflozin Antagonizes Salt‐Sensitive Hypertension Through Modifying Transient Receptor Potential Channels 3 Mediated Vascular Calcium Handling

doi: 10.1161/jaha.121.025328

Figure Lengend Snippet: Figure 3. Canagliflozin inhibited the vascular contraction and calcium channel-related protein pathways. A, Venn diagram in groups between upregulated proteins of ND vs HS and downregulated proteins of HS vs HS+canagliflozin. B, The KEGG pathways enriched by differentially expressed proteins in groups between upregulated proteins of ND vs HS and downregulated proteins of HS vs HS+canagliflozin. C and D, The vascular-associated biological process terms (GO) enriched by differentially expressed proteins proteins in groups between upregulated proteins of ND vs HS and down-regulated proteins of HS vs HS+canagliflozin. E and F, Representative western blots of TRPC3, NCX1, CamkII, MYPT-1, MLC2, and phosphorylation of MYPT-1 and MLC2 (p-MYPT-1 and p-MLC2) in carotid artery from Dahl salt-sensitive and Dahl salt-insensitive rats. β-actin served as a loading control. n=8. Results are expressed as mean±SEM. Statistical significance was determined by unpaired Student t test for comparisons between 2 groups and 1-way ANOVA followed by Bonferron’’s posttest for multiple comparisons. A 2-tailed Fishe’’s exact test was used in the GO KEGG enrichment analyses. *P<0.05, **P<0.01, ***P<0.001. ECM, extracellular matrix; GO, gene ontology; HS, high-salt diet; KEGG, Kyoto Encyclopedia of Genes and Genomes; NCX1, sodium-calcium exchanger 1; ND, normal diet; SS, Dahl salt-sensitive rats; SS-13BN, Dahl salt-insensitive rats; and TRPC3, transient receptor potential channels 3.

Article Snippet: Target antigen Vendor or Source Catalog # Working concentration Lot # (preferred but not required) TRPC3 alomone ACC-016 1:500 NCX1 Abcam ab177952 1:500 CaMKII Abcam ab134041 1:500 Phospho-CaMKII CST 12716S 1:500 MYPT1 Santa Cruz 514261 1:500 Phospho-MYPT1 Santa Cruz 33360 1:500 MLC2 CST 3672 1:500 Phospho- MLC2 CST 3675 1:500 NCX1 Proteintech 55075-1-AP 1:500 D ow nloaded from http://ahajournals.org by on M arch 1, 2024 Figure S1.

Techniques: Western Blot, Phospho-proteomics, Control