Journal: Scientific Reports

Article Title: Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure

doi: 10.1038/s41598-019-42592-1

Figure Lengend Snippet: Tadalafil normalizes the abundance of key regulators of β-adrenergic signalling. ( A ) Representative blot (upper) and summary data (lower) showing no change in β 1 adrenergic receptor abundance with tachypacing or tadalafil treatment. ( B ) Representative blot (upper) and summary data (lower) showing no change in β 2 adrenergic receptor abundance with tachypacing or tadalafil treatment. ( C ) Representative blot (left) and summary data (right) showing tachypacing induced increased and tadalafil mediated normalisation of GRK2 protein abundance. ( D ) Representative blot (upper) and summary data (lower) showing tachypacing induced increased and tadalafil mediated normalisation of PP1 protein abundance. ( E ) Representative blot (upper) and summary data (lower) showing tachypacing induced increased and tadalafil mediated normalisation of PP2A protein abundance. For all panels * P < 0.05 vs. control; # p < 0.05 vs. tadalafil. N = 8 per group.

Article Snippet: Protein phosphatase 1 (PP1) , 20 μg , 10% Milk , 1:1,000, sc-7482 (Santa Cruz Biotechnology) , 1:20,000 IRDye 800CW anti-mouse , Fluorescent.

Techniques: Quantitative Proteomics, Control

Journal: Scientific Reports

Article Title: Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure

doi: 10.1038/s41598-019-42592-1

Figure Lengend Snippet: Blotting conditions for selected proteins.

Article Snippet: Protein phosphatase 1 (PP1) , 20 μg , 10% Milk , 1:1,000, sc-7482 (Santa Cruz Biotechnology) , 1:20,000 IRDye 800CW anti-mouse , Fluorescent.

Techniques:

Journal: American Journal of Human Genetics

Article Title: Bi-allelic KICS2 mutations impair KICSTOR complex-mediated mTORC1 regulation, causing intellectual disability and epilepsy

doi: 10.1016/j.ajhg.2024.12.019

Figure Lengend Snippet: Missense variants do not alter KICS2 levels or influence KICSTOR components, but the p.Lys260Asnfs ∗ 18 variant is ubiquitinated and rapidly degraded by the UPS system (A) Western blot analysis of HEK293T cells overexpressing wild-type (WT) HA-tagged KICS2 or its variants, along with KICSTOR components FLAG-tagged SZT2 and MYC-tagged KPTN, shows a strong reduction in levels of the C-terminally truncated variant p.Lys260Asnfs ∗ 18, while missense variants p.Tyr393Cys and p.Asp296Glu do not affect KICS2 amounts. High-intensity display reveals multiple high-molecular-weight, potentially post-translationally modified forms of KICS2 p.Lys260Asnfs ∗ 18 (KICS2-fs [frameshift], marked with a dashed gray line). Overexpressed proteins were detected using tag-specific antibodies. GAPDH served as the loading control. LI , low intensity; HI , high intensity. (B) Relative quantification of HA-KICS2, FLAG-SZT2, and MYC-KPTN from n = 3 biological replicates ( ∗∗∗ p < 0.001 and ∗ p < 0.05, one-sample t test and two-way ANOVA followed by Tukey’s multiple comparison test). (C–E) Analysis of putatively ubiquitinated (Ub) forms of KICS2 using Ub-Trap immunoprecipitation (IP) of lysates obtained from HEK293T cells transfected with HA-tagged WT KICS2 or KICS2 p.Lys260Asnfs ∗ 18 (KICS2-fs), or an empty control vector (mock), along with MYC-tagged KPTN and FLAG-tagged SZT2. Control beads (ctrl) were used for confirming specificity. Western blot of input samples and eluates, followed by SYPRO Ruby total protein staining, shows successful purification (C). Immunodetection with a Ub-specific antibody shows precipitation of polyubiquitinated (pUb) proteins (marked with a solid black line) (D), while HA-tag-specific immunodetection (E) demonstrates the presence of Ub forms of KICS2-WT and -fs (indicated by solid and dashed gray lines, respectively). GAPDH served as the loading control. The asterisk marks an unspecific protein band present in the input. Unmodified/non-Ub HA-KICS2-WT and -fs were detectable in both the IP:ctrl and IP:Ub lanes, likely due to non-specific or indirect binding of the protein, regardless of its modification status. (F) Investigation of the proteolytic mechanism responsible for the degradation of WT KICS2 and, in particular, the KICS2 p.Lys260Asnfs ∗ 18 (KICS2-fs) variant. HEK293T cells expressing KICS2-WT and KICS2-fs, along with MYC-tagged KPTN and FLAG-tagged SZT2, were treated with the proteasome inhibitor epoxomicin (Epoxo) or autophagy inhibitor bafilomycin A1 (BafA1). DMSO served as the vehicle control. Western blot analysis confirms the accumulation of K48-linked pUb (K48-pUb) chains and LC3B-II as markers of successful proteasomal or autophagosomal inhibition, respectively. HA-tag-specific detection shows a strong accumulation of KICS2-WT and KICS2-fs, as well as their Ub forms (Ub-WT and Ub-fs), upon proteasomal, but not autophagosomal, inhibition. GAPDH served as the loading control. The asterisk marks an unspecific protein band. (G) Relative quantification of KICS2 and Ub KICS2 amounts, relative to vehicle-treated control from n = 3 biological replicates, shows significantly increased levels upon proteasomal inhibition. Calculating the ratios between Ub-KICS2 and KICS2 shows that baseline ubiquitination of KICS-fs is significantly higher compared to KICS2-WT ( ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01, and ∗ p < 0.05, one-sample t test and two-way ANOVA followed by Šídák’s multiple comparison test).

Article Snippet: To inhibit proteasomal or autophagosomal degradation, HEK293T cells were treated with 0.5 μM epoxomicin (BU-4061T, catalog no. S7038) or 50 nM bafilomycin A1 (Baf-A1; catalog no. S1413) (both Selleckchem) 48 h post-transfection.

Techniques: Variant Assay, Western Blot, High Molecular Weight, Modification, Control, Quantitative Proteomics, Comparison, Immunoprecipitation, Transfection, Plasmid Preparation, Staining, Purification, Immunodetection, Binding Assay, Expressing, Inhibition, Ubiquitin Proteomics

Journal: Cell reports

Article Title: Pyruvate metabolism controls chromatin remodeling during CD4 + T cell activation.

doi: 10.1016/j.celrep.2023.112583

Figure Lengend Snippet: Figure 2. ACSS2 inhibition or ACLY deficiency does not affect epigenome remodeling during T cell activation (A) Immunoblots of ACLY, ACSS2, PDCE1, and actin from human T cells activated for 8, 16, 24, or 48 h with anti-CD3/CD28 stimulation. (B and C) CD25 protein expression was measured by flow cytometry (FACS) and IL2ra gene expression was determined by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) in human CD4+ T cells activated with anti-CD3/CD28 in the presence and absence of ACSS2 inhibitor (15.6 mM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER FCCP, mitochondrial oxidative phosphorylation uncoupler Abcam # ab120081 Rotenone Merck Millipore # R8875 UK-5099 (Synonyms: PF-1005023) Med Chem Express # HY-15475 3PO R98% (HPLC) Sigma-Aldrich # SML1343 Sodium acetate Sigma-Aldrich # S1429 DCA Tocris Bioscience # 2755 Etomoxir sodium salt hydrate Sigma-Aldrich # E1905 ACSS2 inhibitor Selleck # S8588 6,8-Bis(benzylthio)-octanoic acid Sigma-Aldrich # SML0404 Sodium Pyruvate Thermo Fisher # 11360070 SB204990 Med Chem Express # HY-16450 BMS303141 Sigma-Aldrich # SML0784 HEPES Sigma-Aldrich #H4034-100G EDTA Sigma-Aldrich #EDS-100G DL-Dithiothreitol solution (DTT) Sigma-Aldrich #43816-10ML Bovine serum albumin (BSA) Sigma-Aldrich #A9647-500G D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML 13C6-glucose Cambridge Isotope Lab #CLM-1396 DMSO Sigma-Aldrich # D2650 Ficoll Paque Plus Sigma-Aldrich # GE17-1440-02 13C6-glucose Cambridge Isotope Lab #CLM-1396 Cell Trace Violet Invitrogen #C34557 Molecular ProbesTM 2-NBDG Invitrogen #N13195 SYBR Thermo fisher #S33102 iScript cDNA kit biorad #170-8891 Formaldehyde Sigma-Aldrich #252549-1L DAPI (1:2000) Invitrogen #D1306 Zombie NIRTM Fixable Viability Kit Biolegend #423106 Zombie GreenTM Fixable Viability Kit Biolegend #423112 7-AAD Invitrogen #A1310 D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML GolgiPlugTM (Protein Transport Inhibitor) BD Biosciences #555029 Tween 20 Sigma-Aldrich #P7949-500ML HALT protease inhibitor Thermo scientific #78439 Powdered milk Carl Roth #T145.2 PierceTM Protein A/G Magnetic Beads thermo fisher #88802 Proteinase K, recombinant, PCR Grade Sigma #3115828001 RNeasy Mini Kit Qiagen #74106 Seahorse XF DMEM medium Agilent Technologies #103575-100 Critical commercial assays MagniSortTM Human CD4+ T cell Enrichment Kit eBioscience #8804-6811-74 CD4 (L3T4) MicroBeads, mouse Miltenyi # 130-117-043 Duolink In Situ Detection Reagents Red Sigma DUO92008 Duolink Green PLA Sigma # DUO92014 Foxp3/Transcription Factor Staining Buffer Set eBioscience #00-5523-00 BD Cytofix/Cytoperm Fixation/Permeabilization kit BD Biosciences #554714 seahorse XFe24 Flux Packs Agilent #1023-40-100 Acetyl-CoA Assay Kit Biovision, Milpitas CA #K317-100 (Continued on next page) Cell Reports 42, 112583, June 27, 2023 19

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Cytometry, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 4: FAIM deficiency suppressed the effects of PACAP on alleviating hepatic lipid accumulation in HFD-fed mice. (A) FAIM protein level of mice liver was subjected to western blot. Statistical analysis of FAIM/b-actin was shown in a bar chart (right) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. CD group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFD group). (B) Schematic diagram of HFD mouse model with PACAP treatment (0.4 mg/kg) or control saline solution and lentivirus mediated Faim knockdown. (C) Hepatic Faim mRNA level was determined by qRT-PCR, n ¼ 3. (DeH) Modulation of Faim level in the liver influenced HFD-induced hepatic steatosis, (D) bodyweight, liver weight, and WAT weight; (E) liver TG and TC contents; (F) plasma TG, TC, HDL-c, and LDL-c levels; (G) fasting blood glucose level; blood glucose concentrations were measured on day 12 after lentivirus injection; (H) representative photomicrographs of Oil Red O and H&E staining of liver tissues in different groups (scale bars: 50 or 200 mM). (I and J) Ablation of Faim impaired glucose tolerance and insulin sensitivity in mice. IPGTT (I) and IPITT (J), blood glucose was measured at different time points after glucose or insulin injection (left), and quantification of the AUC (right). Data are presented as mean SEM, n ¼ 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Western Blot, Control, Saline, Knockdown, Quantitative RT-PCR, Clinical Proteomics, Injection, Staining

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 5: PACAP activates the FAIM-AMPK-IRb axis to govern the SREBP and lipid synthetic gene program. (A and B) PACAP activated AMPK-IRb signaling pathway and down- regulated the expression of lipogenesis genes in HFD-fed mice, such effects were suppressed following knockdown of Faim. Western blot analysis were used to detect the protein level of indicated antibodies. (C and D) The overexpression of FAIM activated AMPK-IRb signaling pathway, the protein levels of FAIM, p-AMPK, AMPK, p-IRb, IRb, and lipid synthesis genes SREBP1, SREBP2, FAS, SCD1, HMGCR were determined by western blot with quantifications. Data are presented as mean SEM, n ¼ 3 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Knockdown, Western Blot, Over Expression

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 6: Knockdown of Faim inhibited the effects of PACAP on decreasing lipid accumulation and activating AMPK-IRb signaling pathway. AML12 cells were transiently transfected with Faim siRNA or scramble siRNA in cell culture medium containing 200 mM PA or 10% BSA (as control of PA solution) for 24 h, then the cells were treated with PACAP (1 mM) and Max.D.4 (10 mM) for another 24 h as indicated. (A) Representative for Oil Red O staining of AML12 (Scale bars, 50 mM). (B and C) Impacts of Faim knockdown on cellular TG and TC level (B), glucose uptake and glycogen content (C) of AML12. (D and E) Faim knockdown impaired the activation of AMPK-IRb pathway in AML12 treated with PACAP, the protein levels were analyzed by western blot and quantitative measurements (right). (F and G) AML12 were exposed to PA for 24 h, then treated with PACAP (1 mM), Max.D.4 (10 mM) and Compound C (10 mM) (F) or AICAR (0.5 mM) (G) for another 24 h. The protein level of FAIM was detected by western blot analysis and quantitative measurements (down). Data are presented as mean SEM. n ¼ 3 (&P < 0.05, &&P < 0.01, &&&P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PA þ PACAP group, OP < 0.05, OOP < 0.01, OOOP < 0.001 vs. PA þ PACAP þ Max.D.4 group).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Knockdown, Transfection, Cell Culture, Control, Staining, Activation Assay, Western Blot

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 7: PACAP activates the PAC1-PKA-CREB signaling pathway to stimulate FAIM expression. (A) mRNA level of Faim in PA-induced AML12 treated with PACAP, determined by qRT-PCR (n ¼ 3). (B) The effect of PACAP on the activation of PKA and CREB in PA induced AML12 cells, the protein levels of PKA, CREB and FAIM, and phosphorylation of PKA and CREB were determined by western blot. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PACAP (1 mM) group). (C) Schematic of the sequence of two CRE binding sites that CREB targets the WT or mutated promoter region of Faim mRNA. (DeF) Luciferase activities in AML12 transiently transfected with either wild type (WT), truncated, or mutant Faim promoter-luciferase reporter constructs. Then the cells were treated with PACAP (1 mM), or co- transfected with CREB cDNA or empty vectors. (G) Luciferase activities of FAIM WT reporter plasmids in AML12 cells with the treatment of KG-501 (a specific CREB inhibitor) and PACAP (1 mM) as indicated. Data are shown as mean SEM, n ¼ 3 or 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Phospho-proteomics, Western Blot, Sequencing, Binding Assay, Luciferase, Transfection, Mutagenesis, Construct

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibit RANKL-induced osteoclastogenesis. A The chemical structural formula of Tetrandrine; B Tetrandrine impact on BMMs viability was detected by CCK-8 assay at 48 h and 96 h. C With different dilutions of Tetrandrine, M-CSF and RANKL for 7 days, BMMs were processed; scale bar = 200 μm. D The number and area of TRAP-positive cells. E TRAP-positive BMMs after the treatment with 1 μM Tetrandrine for the designated days in the process of osteoclastogenesis; scale bar = 200 μm. F To quantify TRAP-positive cells, the number and area of osteoclasts. G Immunofluorescence image for showing the formation of F-actin. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: CCK-8 Assay, Immunofluorescence, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibits osteoclastic bone resportion and expression of osteoclastogenesis related genes. A BMMs were planted into slices of bone and treated with different concentrations of Tetrandrine for 7 days and bone resorption pit pictures taken by a scanning electron microscope are presented; scale bar = 500 μm. The pit zone resorption area were shown by the ImageJ. B The detection of expression levels of osteoclastogenesis related genes in BMMs with 0, 0.25, 0.5, 1 μM was carried out with qPCR. C After treating BMMs with Tetrandrine (1 μM) for 0, 1, 3, 5 days, they were detected via RT-qPCR. The mRNA expression levels of NFATc1, c-Fos, DC-STAMP, TRAP, CTSK were evaluated. D For 0, 1, 3, 5 days with or without 1 μM of Tetrandrine, the treatment of BMMs was carried out with RANKL. The protein expression levels of NFATc1, TRAP and CTSK were evaluated. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Expressing, Microscopy, Quantitative RT-PCR, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine affects osteoclastogenesis by promoting the ubiquitination of TRAIL. A The mRNA expression level of TRAIL in BMMs with 0, 0.25, 0.5, 1 μM of Tetrandrine was carried out with RT-qPCR. B The expression of level of TRAIL in BMMs treated with specified concentrations of Tetrandrine for 5 days was detected using western blot. C Immunofluorescence image for showing TRAIL expression in cytoplasm; scale bar = 200 μm. D The co-IP assay was used to detect the effect of Tetrandrine on TRAIL ubiquitination with or without RANKL. E–G The transfection efficiency of TRAIL silencing (si #1, si #2, si #3) and TRAIL overexpression (TRAIL vector) detected via RT-qPCR and western blotting. H–K BMMs were transfected with siTRAIL and TRAIL vector, the expression of the related genes and proteins that were revealed by RT-qPCR and western blotting. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Ubiquitin Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Co-Immunoprecipitation Assay, Transfection, Over Expression, Plasmid Preparation, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: TRAIL overexpression partially rescued the inhibitory effect of Tetrandrine on osteoclastogenesis. A The mRNA expression of c-Fos, CTSK, DC-STAMP, NFATc1 and TRAP in BMMs in rescue experiment was detected by RT-qPCR. B Western blotting demonstrated the expression of NFATc1, TRAP and CTSK in BMMs in rescue experiment. C The number and area of TRAP-positive cells, the bone resorption pits and F-actin formation in rescue experiment; Scale bar in TRAP staining and immunofluorescence image = 200 μm; scale bar in bone resorption pit picture = 500 μm. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunofluorescence, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibits NF-κB and MAPK pathways by downregulating TRAIL expression. A Treatment of BMMs was carried out with RANKL, with or without 0.25 μM Tetrandrine for 0, 30 and 60 min. The p-P65, p-JNK, p-ERK, p-P38, p-IκBα, p-IKKα/β protein levels were evaluated. B The p-P65, p-JNK, p-P38, p-IκBα, p-IKKα/β protein levels were determined by western blotting in rescue experiment. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Expressing, Western Blot, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine promotes C3H10 osteoblastogenesis and inhibits TRAIL expression in C3H10. A Effect of Tetrandrine on C3H10 viability by CCK-8 assay at 48 h and 96 h. B The TRAIL, OCN, ALP and OPG expression levels in C3H10 treated with Tetrandrine were detected with RT-qPCR. C The protein levels of COL1a, RUNX2, OCN, TRAIL, RANKL and OPG were measured with western blotting. D , E ALP and ARS expression in C3H10 following the treatment with various concentration of Tetrandrine for 7 days. The mineralized extracellular matrix was detected with Alizarin Red S staining for 14 days. The OD values obtained for ALP solutions and mineralized matrix solutions after their treatment with Tetrandrine; scale bar = 200 μm. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Expressing, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Concentration Assay, Staining, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine prevents OVX-induced bone loss in vivo. A Weight change diagram of mice after Tetrandrine treatment (each; n = 5). B RANKL/OPG, TNFα and IL-6 levels in serum of mice were determined with Elisa assay. C , D Detection of the tetrandrine effects on the expression of TRAIL in the OVX-induced model carried out with RT-qPCR and western blot. E Detection of NFATc1, DC-STAMP, TRAP and CTSK expression levels was carried out with RT-qPCR. F Calcein-alizarin red S labeling to detect the femur mineral apposition rate (MAR) in mice. G TRAP staining was used for sections of femurs and number of osteoclasts per field of each specimen were measured. H The Von kossa staining was used for the sections of femurs and the MS/BS (%) was measured. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005 and **** P < 0.001 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Labeling, Staining, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Tetrandrine inhibits OVX-induced osteoporosis in mice model. A H&E staining was used for several major organs of mice in three groups (each; n = 5). B , C The micro-CT was used for the scanning tibias of each mouse, and the BS/BV, Tb.N, Tb.Sp and Tb.Th values were recorded for each specimen. D , E H&E and Masson staining was also used for the femurs. All experiments were carried out in triplicate. The results were presented as mean ± SEM. * P < 0.05 and ** P < 0.01 as compared with the control group

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Staining, Micro-CT, Control

Journal: Molecular Medicine

Article Title: Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL

doi: 10.1186/s10020-022-00568-4

Figure Lengend Snippet: Schematic illustration for the mechanism by which Tetrandrine inhibits RANKL-induced osteoclastogenesis by promoting the degradation of TRAIL. RANKL recruits TRAF6 by interacting with its receptor RANK, and then activates downstream effectors such as NF-κB and MAPKs to promote mature osteoclast formation. Inflammatory factors including IL-6 and TNFα play an active role in the binding of RANK and RANKL and Tetrandrine has obvious anti-inflammatory effect. More importantly, Tetrandrine promotes TRAIL degradation and TRAIL downregulation inhibits the recruitment of TRAF6 by RANKL and eventually dampen osteoclast different and function. Besides, Tetrandrine can stimulate osteoblast difference

Article Snippet: Briefly, 1 × 10 5 cells were added to each well of a 96-well plate and cultivated for 48 and 96 h with Tetrandrine (Selleck, Houston, Texas, United States).

Techniques: Binding Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A : cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. *** P < 0.001. B : this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C : PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. ** P < 0.005. D : PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. * P < 0.05; ** P < 0.005. E : PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. *** P < 0.005. F : PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. *** P < 0.001.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Isolation, Derivative Assay, Inhibition, Thymidine Incorporation Assay, Western Blot, Activation Assay, Proliferation Assay, Positive Control, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Increased expression of p38 MAPK α-isoform in animal models of PH. A : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using the BCA method. Equal concentrations were then loaded on a gel and blotted for p38 MAPKα and β-actin for loading control. There are 3 wells for each condition. Immunoblot shown is best representative of 3 experiments using 3 different animals with each condition. B : densitometry of immunoblot in A . Values are mean arbitrary values from 3 immunoblots expressed relative to the value for β-actin. * P < 0.05 by ANOVA. C : lung sections (5 mm) were prepared from normal, CH, and MCT animals. Sections were stained for p38 MAPKα using 1:400 dilution. Magnification: ×20. Bar represents = 150 mm. Arrows identify blood vessels. D : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then used in a p38 MAPK activity assay using immunoprecipitation and the phosphorylation of activating transcription factor-2 (ATF-2) as a read out. Immunoblot shown is representative of 3 experiments using 3 different animals. E and F : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phosphorylated p38 MAPK and β-actin for loading control. Immunoblot shown is representative of 3 experiments using 3 different animals with each condition. Densitometry is shown for remaining blots. * P < 0.05.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Expressing, Protein Concentration, Control, Western Blot, Staining, Activity Assay, Immunoprecipitation, Phospho-proteomics

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH is prevented by administration of SB203580, a p38 MAPKα inhibitor. A and B : animals were exposed to a hypobaric hypoxic environment for 2 wk. Some animals received daily injections of SB203580. Hemodynamics ( A ) and hematocrit ( B ) were measured after 2 wk. RVSP, right ventricular (RV) systolic pressure. Data represent mean values ± SE. Total animals n = 5–6 per group. C : hearts were isolated from the animals and the RV was dissected out from the left ventricle (LV) and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. The total RV weight was also plotted. Values are means ± SD; n = 5 per group. ** P < 0.05. D : noninvasive systemic blood pressure taken by tail-cuff measurement. E and F : rats were exposed to hypoxia ± SB203580. The lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five random fields per slide were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized and are presented as numbers per total number of vessels present in each field. Groups analyzed by ANOVA for overall change with posttest analysis; n = 5 animals. *** P < 0.001, for A – C and E .

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Isolation, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH in 2 in vivo animal models is reversed by the administration of SB203580, a p38 MAPKα inhibitor. A : animals were exposed to a hypobaric hypoxic environment for 2 wk and then p38 MAPK inhibition was commenced. Hemodynamics and RVSP were measured after 4 wk. Data represent mean values ± SE. Total animals n = 5–6 per group. ** P < 0.01; *** P < 0.001, for normal relative to all other conditions. B : hearts were isolated form the animals and the RV dissected out from the LV and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. Values are means ± SE; n = 6–7. ** P < 0.01; *** P < 0.005. C : hematocrit ratio. D : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. ** P < 0.01; *** P < 0.001. E : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 7 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. ** P < 0.01; *** P < 0.001. F and G : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 6–7 per group. * P < 0.05; *** P < 0.001, for F . * P < 0.05; ** P < 0.01, for G . H : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. * P < 0.05; *** P < 0.001. I : lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 MCT control.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: In Vivo, Inhibition, Isolation, Staining, Control, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH in a reversal strategy in 2 in vivo animal models by administration of PH-797804, a more selective p38 MAPKα inhibitor. A and B : animals were exposed to CH and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RV hypertrophy (RVH) were measured after 4 wk. Data represent mean values ± SE. * P < 0.05; ** P < 0.01 for A . ** P < 0.01; *** P < 0.001, for B . C : the lungs were removed after experiment and sections (5 mm) were cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05 for complete muscularized group in drug-treated vs. day 14 hypoxic control. D : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. E and F : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 14–15 per group. *** P < 0.001 for E . * P < 0.05; ** P < 0.01 for F . G : the lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05, for complete muscularized group in drug-treated vs. day 14 hypoxic control. H : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for drug-treated vs. day 14 MCT control.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: In Vivo, Inhibition, Staining, Control, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: p38 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B : supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. * P < 0.05 ** P < 0.005. C : PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D : PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. ** P < 0.01; *** P < 0.01, for C and D . E : PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. * P < 0.05; ** P < 0.005; *** P < 0.0001. F : PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. **** P < 0.0001. G : PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. ** P < 0.01. H and I : PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. *** P < 0.005; **** P < 0.001, for I .

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, DNA Synthesis, Marker

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A : lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B : lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05 ** P < 0.01. C and D : lungs from CH 28-day controls and SB203580 -treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. ** P < 0.001. E : lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05; ns is not significant by ANOVA. F : PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. *** P < 0.005. G : PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. * P < 0.01; *** P < 0.005. H : fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. ** P < 0.01, *** P < 0.001.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Inhibition, In Vivo, Isolation, Protein Concentration, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Cell Culture, Derivative Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Phospho-p38 MAPK and p38 MAPKα expression in explanted lungs from patients with idiopathic pulmonary arterial hypertension (IPAH). A : sections of 5 mm were taken. Then, normal control lung and IPAH lung are stained for phospho-p38 MAPK at dilution of 1:300. The isotype on IPAH lung is also shown. This dilution was optimally assessed for. Objective: ×20. Bar = 150 mm. B : high-power microscopy shows that there is strong staining for phospho-p38 MAPK in the intima, media, and the adventitia (arrows). Objective lens: ×20 and ×40. Bar = 50 mm. C : sections of control lung ( A ) and IPAH lung ( B ) were stained for p38 MAPKα at dilution of 1:300. Objective lens × 20. Bar = 150 mm. D : staining for p38 MAPKa showed increased cytosolic staining in the IPAH lung ( right ) compared with control lung ( left ). E : high-power view (×40) of staining with isotype and p38 MAPKα in a vessel in IPAH lung. This shows staining throughout the vessel layers but especially in adventitia and fibroblast cells (arrow). Bar = 50 mm. F : low-power view of a plexiform lesion. Staining for p38 MAPKα using 1:300 dilution. Objective lens × 20. Bar = 150 mm. G : histological scoring shows increased p38 MAPKα staining throughout the vascular wall. With the use of a well-validated histological scoring system (Allred), the vascular wall cells were scored for intensity of staining. The intensity multiplied by the number of vessels with that intensity determines the values. Values shown are from 5 random high-power fields from 2 slides. **** P < 0.0001 by ANOVA. ** P < 0.001 for individual IPAH vs. control columns.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Expressing, Control, Staining, Microscopy

Journal: Scientific Reports

Article Title: Microbial metabolite butyrate facilitates M2 macrophage polarization and function

doi: 10.1038/srep24838

Figure Lengend Snippet: ( a ) Arg1 and Ym1 gene expression in butyrate-treated M2 macrophage treated with increasing concentrations (2–20 μM) of HAT inhibitor, C646. ( b ) Arg1 and Ym1 gene expression in the presence of butyrate (50 μg/ml) and C646 (5 μM). ( c ) Western blot analysis of Arg1 protein in butyrate-treated M2-BMDMs after 12 h treatment with C646 (5 μM). ( d ) Quantitative RT-PCR analysis of HDAC1 gene expression in butyrate-treated M2-BMDMs after 6 h treatment with C646 (5 μM). ( e ) Western blot analysis of acetylated H3K9, total histone H3, and β-actin proteins in butyrate-treated M2-BMDMs after 12 h treatment with C646 (5 μM). Cells were lysed and western blotting performed with the indicated antibodies. ( f ) Phosphorylation of STAT6 in butyrate-treated M2-BMDMs. Western blotting was performed with anti-phospho-STAT6, STAT6, and β-actin. Data are representative of three independent experiments.

Article Snippet: The BMDMs were subsequently cultured for 1.5-24 h in the presence of sodium butyrate (Sigma-Aldrich) or histone acetyltransferase (HAT) inhibitor C646 (Selleck).

Techniques: Gene Expression, Western Blot, Quantitative RT-PCR, Phospho-proteomics

Journal: bioRxiv

Article Title: FUNGAL DYSBIOSIS CORRELATES WITH THE DEVELOPMENT OF TUMOUR-INDUCED CACHEXIA IN MICE

doi: 10.1101/2020.06.29.171397

Figure Lengend Snippet: Equimolar samples of DNA extracted from stool samples obtained from each mouse were consolidated into two pools, representing SC and CC groups. Samples from these pools were then used in qPCR experiments, with primers previously described in the literature as specific to Saccharomyceae, Kazachstania and Malassezia genera, as well as to the species Saccharomyces cereviseae, Malassezia dermatis, Malassezia japonica, Rhizopus oryzeae and Penicillium citrinum . The Ct values obtained for each of these taxa were normalized by the Ct obtained after amplifying the pooled DNAs with the primer pair used to amplify the fungal ITS1 amplicons . Next, the normalized Ct s were used to calculate the relative prevalence of each genus/species in the CC pool, in relation to the SC pool (CC/CR). The relative quantification values for each taxon ( Fold Change ) are shown in the graph, as the mean ± standard deviation of three independent experiments (each performed in triplicate). The species Rhizopus oryzae was detected only in the DNA pool from SC animals, so its SC/CC ratio is represented in the graph as negative, to infinity (-INF). The species P enicillium citrinum was not detected (N/D) in any of the experiments, with either DNA pool.

Article Snippet: Average sizes of the generated fragments were evaluated after electrophoresis in a Bioanalyzer 2100 (Agilent), using the High Sensitivity DNA Chip and the libraries were quantified by qPCR, in an ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems), with the aid of the universal quantification kit NEBNext (New England Biolabs), according to the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Standard Deviation