Journal: bioRxiv

Article Title: Super Enhancer-driven LncRNA UNC5B-AS1 Inhibits Inflammatory Phenotypic Transition in Smooth Muscle Cells via Lactylation Modification

doi: 10.1101/2024.05.07.593065

Figure Lengend Snippet: A Schematic diagram of the SU5416 combined with hypoxia-induced PH (SuHx) mice model. B Expression of UNC5B-AS1 conserved fragment (UA1CF) in mice lungs (n=8). C Right ventricle/left ventricle + septum (RV/LV+ Septum) in mice (n=8). D Representative waveform of right ventricular systolic pressure. E Representative right ventricular systolic pressure (RVSP) in mice (n=8). Pulmonary artery acceleration time (PAAT, F ), pulmonary artery velocity-time integral (PAVTI, G ), ejection fraction (EF, H ) in mice (n=8). I Representative images of Hematoxylin and Eosin staining (H&E) and Masson staining in mice lungs. Scale bars=50 μm. All data are presented as mean ± SEM. One-way ANOVA with Tukey post hoc test was used to compare multiple groups with equal variance, and Brown-Forsythe and Welch ANOVA with Tamhane T2 post hoc test was used to compare multiple groups with unequal variance. Nor, normoxia, SuHx, SU5416 combined with hypoxia, NC, Negative control and UA1CF, AAV5-UNC5B-AS1 conserved fragment overexpression.

Article Snippet: In the Sugen hypoxia model, the mice were injected once per week with the VEGF inhibitor SU5416 (Med Chem Express, USA) at a concentration of 20 mg/kg.

Techniques: Expressing, Staining, Negative Control, Over Expression

Journal: Cell Death & Disease

Article Title: Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

doi: 10.1038/cddis.2013.192

Figure Lengend Snippet: Killing of BRAF V600E melanoma cells by cotreatment with SAHA and PLX4720 is associated with activation of the caspase cascade and damage to the mitochondria. ( a ) HEMn-MP melanocytes, Sk-Mel-28, and Mel-CV melanoma cells treated with the vehicle control (DMSO), SAHA (2 μ M), PLX4720 (5 μ M), or the combination of SAHA and PLX4720 for 48 h were subjected to CellTiter-Glo assays. The data shown are mean±S.E.M. of three individual experiments. * P <0.01, two-tailed Student's t -test. ( b ) Upper panel: MM200 and Sk-Mel-28 cells were cotreated with SAHA (2 μ M) and PLX4720 (5 μ M) for indicated periods. Induction of cell death was quantitated by the Annexin V-fluorescein isothiocyante (FITC)/propidium iodide (PI) method. The number in each right bottom quadrant represents the percentage of viable cells in each sample. Lower panel: Comparison of the proportion of dead cells with PI uptake and the proportion of dead cells negative for PI as shown in the upper panel. The data shown are representative of three individual experiments. ( c ) Upper panel: MM200 and Sk-Mel-28 cells treated with the vehicle control (DMSO) or the combination of SAHA (2 μ M) and PLX4720 (5 μ M) for 36 h were subjected to measurement of reduction in the mitochondrial potential using JC-1 staining. The number in each bottom-left quadrant represents the percentage of cells with reduction in the mitochondrial potential. Lower panel: MM200 and Sk-Mel-28 cells were treated with SAHA (2 μ M), PLX4720 (5 μ M), or the combination of both for 36 h. Cytosolic and mitochondrial fractions were subjected to western blot analysis of cytochrome C and Smac/DIABLO. Analysis of β -actin and COX IV were included for relative purity of cytosolic and mitochondrial fractions, respectively. The data shown are representative of three individual experiments. ( d ) MM200 and Sk-Mel-28 cells were treated with SAHA (2 μ M), PLX4720 (5 μ M), or the combination of both for 48 h. Whole-cell lysates were subjected to western blot analysis of caspase-3, caspase-9, the 89 kDa fragment of cleaved PARP (using an antibody that specifically recognizes this fragment), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (as a loading control). The data shown are representative of three individual experiments. ( e ) MM200 and Sk-Mel-28 cells were seeded at 1000 cells per well onto 6-well plates as single-cell suspension. After 24 h, SAHA (2 μ M), PLX4720 (5 μ M), or the combination of both was added into the culture medium. Cells were allowed to grow for 12 days before being fixed with methanol and stained with crystal violet. The data shown are representative of three individual experiments. ( f ) Whole-cell lysates from MM200 and Sk-Mel-28 cells treated with SAHA (2 μ M), PLX4720 (5 μ M), or the combination of both for 3 h were subjected to western blot analysis of phosphorylated ERK1/2 (pERK1/2), ERK1/2, and GAPDH (as a loading control). The data shown are representative of three individual experiments

Article Snippet: The cell-permeable general caspase inhibitor z-VAD-fmk was purchased from Calbiochem (La Jolla, CA, USA).

Techniques: Activation Assay, Control, Two Tailed Test, Comparison, Staining, Western Blot, Suspension

Journal: Cell Death & Disease

Article Title: Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

doi: 10.1038/cddis.2013.192

Figure Lengend Snippet: Induction of cell death by combinations of SAHA and PLX4720 is largely independent of the caspase cascade. ( a ) MM200, Sk-Mel-28, IgR3, and Mel-RMu cells with or without pretreatment with z-VAD-fmk (30 μ M) for 1 h were treated with SAHA (2 μ M), PLX4720 (5 μ M), or the combination of both for a further 48 h. MM200 and Mel-RMu cells treated with TNF-related apoptosis-inducing ligand (TRAIL) (200 ng/ml) with or without pretreatment with z-VAD-fmk were included as controls. Cell viability was measured by CellTiter-Glo assays. The data shown are mean±S.E.M. of three individual experiments. * P <0.01, two-tailed Student's t -test. ( b ) MM200, Sk-Mel-28, IgR3, and Mel-RMu cells were transfected with the control or caspase-3 siRNA. After 24 h, whole-cell lysates were subjected to western blot analysis of caspase-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (as a loading control). The data shown are representative of three individual experiments. ( c ) MM200, Sk-Mel-28, IgR3, and Mel-RMu cells were transfected with the control or caspase-3 siRNA. After 24 h, cells were treated with SAHA (2 μ M), PLX4720 (5 μ M), or the combination of both for a further 48 h. Cell viability was measured by CellTiter-Glo assays. The data shown are mean±S.E.M. of three individual experiments. * P <0.01, two-tailed Student's t -test

Article Snippet: The cell-permeable general caspase inhibitor z-VAD-fmk was purchased from Calbiochem (La Jolla, CA, USA).

Techniques: Two Tailed Test, Transfection, Control, Western Blot

Journal: Cell Death & Disease

Article Title: Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

doi: 10.1038/cddis.2013.192

Figure Lengend Snippet: Cotreatment with SAHA and vemurafenib inhibits melanoma xenograft growth in a mouse model. ( a ) MM200 (left) and Sk-Mel-28 (right) cells (1 × 10 7 ) were xenografted into flanks of nu/nu mice viasubcutaneous injection. Ten days after transplantation when xenografts reached approximately 100 mm 3 , mice were administered with either the vehicle (DMSO) ( n =8) or SAHA (100 mg/kg per day) ( n =8) via intraperitoneal injection, vemurafenib (75 mg/kg per day) ( n =8) via oral gavage, or the combination of SAHA and vemurafenib daily for 10 days. Mice were euthanized at 28 days after melanoma cell injection. The data shown are growth curves of melanoma tumors represented by the volume calculated with the modified ellipsoidal formula (tumor volume=1/2(length × width 2 )), which are mean±S.E.M. of all tumors in each experimental group (* P <0.001, Student's t -test). ( b ) Whole-cell lysates from MM200 cells transduced with the control or caspase-3 short hairpin RNA (shRNA) were subjected to western blot analysis of caspase-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (as a loading control). The data shown are representative of three individual western blots. ( c ) MM200 cells transduced with the control or caspase-3 shRNA were xenografted into flanks of nu/nu mice via subutaneous injection. Ten days after transplantation, mice were administered with either the vehicles ( n =8) or SAHA (100 mg/kg per day) ( n =8) via intraperitoneal injections, vemurafenib (75 mg/kg per day) ( n =8) via oral gavage, or the combination of SAHA and vemurafenib daily for 10 days. Mice were euthanized at 28 days after melanoma cell injection. The data shown are tumor volume at the date of euthanization, which are mean±S.E.M. of all tumors in each experimental group (* P <0.001, Student's t -test). ( d ) Whole-cell lysates of crude tumor tissues randomly sampled from tumors formed with MM200 cells transduced with the control shRNA before and undergoing treatment with SAHA in combination with vemurafenib were subjected to western blot analysis of caspase-3 and GAPDH (as a loading control). The data shown are representative of three individual experiments

Article Snippet: The cell-permeable general caspase inhibitor z-VAD-fmk was purchased from Calbiochem (La Jolla, CA, USA).

Techniques: Injection, Transplantation Assay, Modification, Transduction, Control, shRNA, Western Blot

Journal: Current Issues in Molecular Biology

Article Title: Pro-Angiogenetic Effects of Purified Extracts from Helix aspersa during Zebrafish Development

doi: 10.3390/cimb44080232

Figure Lengend Snippet: Pretreatment with a VEGF pathway inhibitor prevented the formation of intersomitic vessels: The panels show representative images of a WISH assay performed with fli1 as an endothelial marker, at 36 hpf, in untreated ( A , C , E , G , I ) and treated embryos ( B , D , F , H , J ) with SUGEN 5416. Previously, some embryos were treated with snail derivatives via Method I and controls with fish water and 0.1% DMSO (see Materials and Methods, and ). Magnification of the trunk region (32×). Two replicates were performed ( n = 15). Ratios at the bottom-left part of each picture specify the number of embryos showing the same staining pattern, compared to the total number of embryos used for each experiment. The images were taken in lateral position at 32× magnification with a Zeiss Axiozoom V13 (Zeiss, Jena, Germany) microscope, equipped with a PlanNeoFluar Z 1×/0.25 FWD 56 mm lens and Zen Pro software.

Article Snippet: Zebrafish embryos were then treated at 4 hpf with the snail derivatives LH, LM, LH3, and LM2, and then at 15 hpf with the VEGF inhibitor SU5416 (Sunitinib, Merck KGaA, Darmstadt, Germany) at a final concentration of 5 μg/mL in fish water and incubated in 3 cm diameter well-plates.

Techniques: Marker, Staining, Microscopy, Software

Journal: Scientific reports

Article Title: Beneficial effect of heat-killed Lactiplantibacillus plantarum L-137 on intestinal barrier function of rat small intestinal epithelial cells.

doi: 10.1038/s41598-024-62657-0

Figure Lengend Snippet: Figure 5. Effect of specific pathway inhibitors (ERK1/2; PD98059, JNK; SP600125, p38; SB203580, AMPK; dorsomorphin, Akt; LY294002) on improving FD-4 permeability of IEC-6 cells by HK L-137. FD-4 was added to rat IEC-6 cells treated with (A) PD98059 (25 μM), (B) SP600125 (10 μM), (C) SB203580 (10 μM), (D) dorsomorphin (20 μM), and (E) LY294002 (5 μM) for 1 h and then with HK L-137 (500 μg/mL) for 26 h, and fluorescence intensity was measured 5 h later. Excitation wavelength, 490 nm; emission wavelength, 520 nm. Means ± SD; n = 3 (B,C) or 4 (A,D,E), Newman–Keuls test, *P < 0.05, **P < 0.01, ***P < 0.001. DO dorsomorphin, FD-4 fluorescein isothiocyanate-dextran, HK L-137 heat-killed Lactiplantibacillus plantarum L-137, LY LY294002, PD PD98059, SP SP600125, SB SB203580.

Article Snippet: Chemicals were purchased from various suppliers, as follows: Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, Bovine insulin, fluorescein isothiocyanate-dextran (FD-4), complete protease inhibitor cocktail tablets, and PhosStop phosphatase inhibitor tablets, from Sigma-Aldrich (Saint Louis, MO, USA); sodium hydrogen carbonate, radioimmunoprecipitation assay (RIPA) buffer, PD98059, dorsomorphin, paraformaldehyde, polyoxyethylene (10) octylphenyl ether (TritonX-100), from Fujifilm Wako (Osaka, Japan); fetal bovine serum (FBS), from HyClone (Logan, UT, USA); primary anti-mouse antibodies for Akt (pan), primary anti-rabbit antibodies for phospho-Akt (Ser473), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), LY294002, SP600125, SB203580, from Cell Signaling Technology (Danvers, MA, USA); antimouse occludin antibody, anti-rabbit ZO-1 antibody, donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody-alexa fluor 488 (secondary fluorescent dye-conjugated donkey anti-rabbit IgG), from Invitrogen (Waltham, MA, USA); horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, HRPconjugated anti-rabbit secondary antibody from Protein Simple (San Jose, CA, USA); Cellstain®-DAPI, from DOJINDO LABORATORIES (Kumamoto, Japan); anti-rabbit ZO-1 antibody, from Proteintech Group (Rosemont, IL, USA); FluoroQuestTM Anti-fading Kit II (antifade agent), from AAT Bioquest (Pleasanton, CA, USA); normal donkey serum, from Jackson Immuno Research Laboratories (West Grove, PA, USA); and phosphate buffered saline (PBS) tablets, from Takara (Shiga, Japan).

Techniques: Permeability, Fluorescence

Journal: Scientific reports

Article Title: Beneficial effect of heat-killed Lactiplantibacillus plantarum L-137 on intestinal barrier function of rat small intestinal epithelial cells.

doi: 10.1038/s41598-024-62657-0

Figure Lengend Snippet: Figure 6. Effect of HK L-137 on phosphorylated protein expression levels in IEC-6 cells. (A) p-ERK/ERK, (B) p-AMPK/AMPK, (C) p-Akt/Akt levels were measured in rat IEC-6 cells treated with or without the inhibitors of ERK1/2 (PD98059, 25 μM), AMPK (dorsomorphin, 20 μM), or Akt (LY294002, 5 μM) for 1 h, followed by 15 min of treatment with HK L-137 (500 μg/mL). Images of representative bands are shown. Full-length images are shown in Supplemental Fig. S2. P-ERK, p-AMPK, and p-Akt expression levels were corrected for ERK, AMPK, and Akt expression levels. The expression levels of ERK and p-ERK were calculated by adding the area values of 42 kDa and 44 kDa. Means ± SD; n = 3, Newman–Keuls test, **P < 0.01, ***P < 0.001. AMPK AMP- activated protein kinase, DO dorsomorphin, ERK extracellular signal-regulated kinase, HK L-137 heat-killed Lactiplantibacillus plantarum L-137, LY LY294002, PD PD98059, p-AMPK phosphorylated AMP-activated protein kinase, p-ERK phosphorylated extracellular signal-regulated kinase.

Article Snippet: Chemicals were purchased from various suppliers, as follows: Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, Bovine insulin, fluorescein isothiocyanate-dextran (FD-4), complete protease inhibitor cocktail tablets, and PhosStop phosphatase inhibitor tablets, from Sigma-Aldrich (Saint Louis, MO, USA); sodium hydrogen carbonate, radioimmunoprecipitation assay (RIPA) buffer, PD98059, dorsomorphin, paraformaldehyde, polyoxyethylene (10) octylphenyl ether (TritonX-100), from Fujifilm Wako (Osaka, Japan); fetal bovine serum (FBS), from HyClone (Logan, UT, USA); primary anti-mouse antibodies for Akt (pan), primary anti-rabbit antibodies for phospho-Akt (Ser473), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), LY294002, SP600125, SB203580, from Cell Signaling Technology (Danvers, MA, USA); antimouse occludin antibody, anti-rabbit ZO-1 antibody, donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody-alexa fluor 488 (secondary fluorescent dye-conjugated donkey anti-rabbit IgG), from Invitrogen (Waltham, MA, USA); horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody, HRPconjugated anti-rabbit secondary antibody from Protein Simple (San Jose, CA, USA); Cellstain®-DAPI, from DOJINDO LABORATORIES (Kumamoto, Japan); anti-rabbit ZO-1 antibody, from Proteintech Group (Rosemont, IL, USA); FluoroQuestTM Anti-fading Kit II (antifade agent), from AAT Bioquest (Pleasanton, CA, USA); normal donkey serum, from Jackson Immuno Research Laboratories (West Grove, PA, USA); and phosphate buffered saline (PBS) tablets, from Takara (Shiga, Japan).

Techniques: Expressing