Journal: Communications Biology

Article Title: Farnesoid X receptor activation inhibits TGFBR1/TAK1-mediated vascular inflammation and calcification via miR-135a-5p

doi: 10.1038/s42003-020-1058-2

Figure Lengend Snippet: HASMCs were cultured with osteogenic medium containing 2.0 mM Pi and 2.7 mM Ca. After 7 days, cells were stained with alizarin red (100×) a which identify calcium mineral as red. b Ca content assayed by calcium detection assay kit, which was normalized to protein concentration. c The mRNA expression of TGFBR1, TAK1, TAB1, and IκBα in osteogenic medium-treated HASMCs. d Western blot analysis of the expression of TAK1, p-TAK1, TAB1, and p-IκBα in osteogenic medium-treated HASMCs. e Western blot analysis of the expression of NF-κB, TNF-α, TAK1, p-TAK1, TAB1, and p-IκBα in osteogenic medium-treated HASMCs in the absence or presence of TGFBR1 siRNA. f Representative immunofluorescence staining of NF-κB (green) and TNF-α (red) in indicated groups (100×). g Calcium content of HASMCs in indicated groups. h Representative images of alizarin red staining in indicated groups (100×). * P < 0.05, ** P < 0.01. ns indicates not significant. Data were pooled as mean ± S.D. (error bars) from three or more independent experiments.

Article Snippet: TaqMan RT-PCR was performed in triplicate using the ViiA TM 7 Real-Time PCR System (Applied Biosystems) with the following probes (Life Technologies): 478581_mir (hsa-miR-135a-5p/rno-miR-135a-5p), Hs00610320_m1 (human TGFBR1), Rn00688966_m1 (rat tgfbr1), Hs00751239_s1 (human Msx2), Hs01047973_m1 (human Runx2), Hs01866874_s1 (human Osterix/SP7), Hs01029144_m1 (human ALP), Hs00959010_m1 (human OPN/SPP1), Hs01587814_g1 (human BGLAP/OCN), Hs06633309_s1 (human NF-κB), Hs02621508_s1 (human TNF-α), Hs00174131_m1 (human IL-6), Hs00196143_m1 (human TAB1), Rn01504766_m1 (rat TAB1), Hs00355671_g1 (human NFKBIA/IκBα), Rn01473657_g1 (rat Nfkbia /IκBα), Hs00177373_m1 (human MAP3K7/TAK1), and Rn01437015_m1 (rat Map3k7/TAK1).

Techniques: Cell Culture, Staining, Detection Assay, Protein Concentration, Expressing, Western Blot, Immunofluorescence

Journal: Communications Biology

Article Title: Farnesoid X receptor activation inhibits TGFBR1/TAK1-mediated vascular inflammation and calcification via miR-135a-5p

doi: 10.1038/s42003-020-1058-2

Figure Lengend Snippet: Wistar rats were fed either chow diet or high-phosphate diet in the absence or presence of 0.75% adenine to induce chronic kidney disease for 4 weeks, all rats were fed for another 4 weeks to form vascular calcification ( n = 8 per group). a Representative images of alizarin red staining and von Kossa staining of aortic roots or aortic ring in indicated groups (scale bar, 500 μm). b Calcified lesion areas of aortic in indicated groups. c Aortic calcium content analysis in indicated groups. d Representative images of micro-CT, arrows indicate calcified nodules. e Representative immunofluorescence staining of NF-κB (red) and TNF-α (green) of aortic roots in indicated groups (scale bar, 500 μm). f Western blot analysis of NF-κB and TNF-α expression in aortas in indicated groups. g Western blot analysis of the expression of TAK1, p-TAK1, TAB1, and p-IκBα in high-adenine diet-fed rats (HAD group, n = 8 per group). ** P < 0.01, ns indicates not significant.

Article Snippet: TaqMan RT-PCR was performed in triplicate using the ViiA TM 7 Real-Time PCR System (Applied Biosystems) with the following probes (Life Technologies): 478581_mir (hsa-miR-135a-5p/rno-miR-135a-5p), Hs00610320_m1 (human TGFBR1), Rn00688966_m1 (rat tgfbr1), Hs00751239_s1 (human Msx2), Hs01047973_m1 (human Runx2), Hs01866874_s1 (human Osterix/SP7), Hs01029144_m1 (human ALP), Hs00959010_m1 (human OPN/SPP1), Hs01587814_g1 (human BGLAP/OCN), Hs06633309_s1 (human NF-κB), Hs02621508_s1 (human TNF-α), Hs00174131_m1 (human IL-6), Hs00196143_m1 (human TAB1), Rn01504766_m1 (rat TAB1), Hs00355671_g1 (human NFKBIA/IκBα), Rn01473657_g1 (rat Nfkbia /IκBα), Hs00177373_m1 (human MAP3K7/TAK1), and Rn01437015_m1 (rat Map3k7/TAK1).

Techniques: Staining, Micro-CT, Immunofluorescence, Western Blot, Expressing

Journal: Communications Biology

Article Title: Farnesoid X receptor activation inhibits TGFBR1/TAK1-mediated vascular inflammation and calcification via miR-135a-5p

doi: 10.1038/s42003-020-1058-2

Figure Lengend Snippet: HASMCs were cultured with osteogenic medium in the absence or presence of 3 μM OCA (FXR ligand) for 7 days. a Western blot results showed that FXR was activated by OCA. b Western blot analysis of the expression of TAK1, p-TAK1, TAB1, p-IκBα, NF-κB, and TNF-α in indicated groups. c Representative immunofluorescence staining of NF-κB (green) and TNF-α (red) in indicated groups (100×). d Intercellular NF-κB content of HASMCs in indicated groups. e Representative images of alizarin red staining in indicated groups (100×). f Calcium content of HASMCs in indicated groups. g The mRNA expression of Runx2 and ALP in indicated groups. * P < 0.05, ** P < 0.01. Data were pooled as mean ± S.D. (error bars) from three or more independent experiments.

Article Snippet: TaqMan RT-PCR was performed in triplicate using the ViiA TM 7 Real-Time PCR System (Applied Biosystems) with the following probes (Life Technologies): 478581_mir (hsa-miR-135a-5p/rno-miR-135a-5p), Hs00610320_m1 (human TGFBR1), Rn00688966_m1 (rat tgfbr1), Hs00751239_s1 (human Msx2), Hs01047973_m1 (human Runx2), Hs01866874_s1 (human Osterix/SP7), Hs01029144_m1 (human ALP), Hs00959010_m1 (human OPN/SPP1), Hs01587814_g1 (human BGLAP/OCN), Hs06633309_s1 (human NF-κB), Hs02621508_s1 (human TNF-α), Hs00174131_m1 (human IL-6), Hs00196143_m1 (human TAB1), Rn01504766_m1 (rat TAB1), Hs00355671_g1 (human NFKBIA/IκBα), Rn01473657_g1 (rat Nfkbia /IκBα), Hs00177373_m1 (human MAP3K7/TAK1), and Rn01437015_m1 (rat Map3k7/TAK1).

Techniques: Cell Culture, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Communications Biology

Article Title: Farnesoid X receptor activation inhibits TGFBR1/TAK1-mediated vascular inflammation and calcification via miR-135a-5p

doi: 10.1038/s42003-020-1058-2

Figure Lengend Snippet: To inhibit miR135a-5p, rats were injected with antagomiR135a-5p (80 μg/g body weight) through vena caudalis for three consecutive days before inducing chronic kidney disease ( n = 8 per group). a The mRNA expression of TGFBR1 of aortas in indicated groups. b , c Calcified lesion areas of aortic ( b ) and aortic calcium content ( c ) in indicated groups. d Representative images of von Kossa staining of aortic ring in indicated groups (scale bar, 500 μm). e Representative images of micro-CT in indicated groups, arrows indicate calcified nodules. f Western blot analysis of NF-κB, TNF-α, TAK1, p-TAK1, TAB1, and p-IκBα of aortas in indicated groups. g Representative immunofluorescence staining of p-IκBα (pink), TAK1 (red) and TAB1 (green) of aortic rings in indicated groups (scale bar, 500μm). * P < 0.05, ** P < 0.01, ns indicates not significant.

Article Snippet: TaqMan RT-PCR was performed in triplicate using the ViiA TM 7 Real-Time PCR System (Applied Biosystems) with the following probes (Life Technologies): 478581_mir (hsa-miR-135a-5p/rno-miR-135a-5p), Hs00610320_m1 (human TGFBR1), Rn00688966_m1 (rat tgfbr1), Hs00751239_s1 (human Msx2), Hs01047973_m1 (human Runx2), Hs01866874_s1 (human Osterix/SP7), Hs01029144_m1 (human ALP), Hs00959010_m1 (human OPN/SPP1), Hs01587814_g1 (human BGLAP/OCN), Hs06633309_s1 (human NF-κB), Hs02621508_s1 (human TNF-α), Hs00174131_m1 (human IL-6), Hs00196143_m1 (human TAB1), Rn01504766_m1 (rat TAB1), Hs00355671_g1 (human NFKBIA/IκBα), Rn01473657_g1 (rat Nfkbia /IκBα), Hs00177373_m1 (human MAP3K7/TAK1), and Rn01437015_m1 (rat Map3k7/TAK1).

Techniques: Injection, Expressing, Staining, Micro-CT, Western Blot, Immunofluorescence

Journal: Communications Biology

Article Title: Farnesoid X receptor activation inhibits TGFBR1/TAK1-mediated vascular inflammation and calcification via miR-135a-5p

doi: 10.1038/s42003-020-1058-2

Figure Lengend Snippet: a TGFBR1 is activated in osteogenic medium-cultured HASMCs, and then activates TAK1, which binds with TAB1 and TAB2 to form a stable complex at the N-terminal kinase domain and the C-terminal region of TAK1. NF-κB pathway is the downstream of TAK1, which is reported to stimulate the expression of osteogenic transcription factor and osteogenic marker, resulting in calcification formation of HASMCs. MicroRNA-135a-5p is key regulator to inhibited TGFBR1, which is suppressed in osteogenic medium-cultured HASMCs. b FXR activation increases microRNA-135a-5p expression to inhibit TGFBR1/TAK1 pathway and further attenuates osteogenic medium-induced HASMCs inflammation and calcification.

Article Snippet: TaqMan RT-PCR was performed in triplicate using the ViiA TM 7 Real-Time PCR System (Applied Biosystems) with the following probes (Life Technologies): 478581_mir (hsa-miR-135a-5p/rno-miR-135a-5p), Hs00610320_m1 (human TGFBR1), Rn00688966_m1 (rat tgfbr1), Hs00751239_s1 (human Msx2), Hs01047973_m1 (human Runx2), Hs01866874_s1 (human Osterix/SP7), Hs01029144_m1 (human ALP), Hs00959010_m1 (human OPN/SPP1), Hs01587814_g1 (human BGLAP/OCN), Hs06633309_s1 (human NF-κB), Hs02621508_s1 (human TNF-α), Hs00174131_m1 (human IL-6), Hs00196143_m1 (human TAB1), Rn01504766_m1 (rat TAB1), Hs00355671_g1 (human NFKBIA/IκBα), Rn01473657_g1 (rat Nfkbia /IκBα), Hs00177373_m1 (human MAP3K7/TAK1), and Rn01437015_m1 (rat Map3k7/TAK1).

Techniques: Cell Culture, Expressing, Marker, Activation Assay

Journal: Molecular and Cellular Biochemistry

Article Title: The inflammatory signalling mediator TAK1 mediates lymphocyte recruitment to lipopolysaccharide-activated murine mesenchymal stem cells through interleukin-6

doi: 10.1007/s11010-021-04180-8

Figure Lengend Snippet: Stimulation of native murine MSCs with 1 µg/ml LPS for different time points. Western blot analyses for protein kinases involved in LPS signal transduction were performed as detailed in . Note that the molecular weight of the bands detected with the different phospho-TAK1 antibodies is lower than the molecular weight of total TAK1, probably arising from non-specific staining

Article Snippet: The gene-specific assays as well as the Fast Advanced Mastermix were purchased from Life Technologies: Rps29 Mm02342448_gH (housekeeping gene), Tak1 (Map3k7) Mm00554514_m1.

Techniques: Western Blot, Transduction, Molecular Weight, Staining

Journal: Molecular and Cellular Biochemistry

Article Title: The inflammatory signalling mediator TAK1 mediates lymphocyte recruitment to lipopolysaccharide-activated murine mesenchymal stem cells through interleukin-6

doi: 10.1007/s11010-021-04180-8

Figure Lengend Snippet: TAK1 downregulation by lentiviral-mediated shTAK1 expression in mMSCs affects LPS-stimulated signalling pathways. After lentiviral modulation of TAK1 expression inflammatory signalling pathways were assessed after LPS stimulation (LPS: 1 µg/ml, 30 min). (A) p38, IκB and JNK pathways were analysed after Western blotting with antibodies specific for activated signalling factors. One representative result of three individual experiments is shown. (B-F) The graphs demonstrate densitometric analysis of TAK1 (B) and JNK (C and D) , p-p38 (E) and IκBα pathways (F) , mean ± SEM. The average density level of each band from three individual experiments was corrected for actin loading controls. Densitometric analysis of blots was performed with ImageJ 1.53c following the published protocol . For the pairwise comparisons the Students t-test was used *: p < 0.05, **: p < 0.01, ***: p < 0.001. (G) Quantification of TAK1 expression by quantitative real-time PCR in the four experimental groups, three independent experiments. Relative gene expression analysis (2 −ΔCt ), mean ± SEM. (H) The data from (C) were set to 100% and the downregulation of TAK1 expression was calculated for the non-stimulated samples and the LPS-stimulated samples, respectively

Article Snippet: The gene-specific assays as well as the Fast Advanced Mastermix were purchased from Life Technologies: Rps29 Mm02342448_gH (housekeeping gene), Tak1 (Map3k7) Mm00554514_m1.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Molecular and Cellular Biochemistry

Article Title: The inflammatory signalling mediator TAK1 mediates lymphocyte recruitment to lipopolysaccharide-activated murine mesenchymal stem cells through interleukin-6

doi: 10.1007/s11010-021-04180-8

Figure Lengend Snippet: Lymphocyte recruitment by TAK1 genetically modified mMSCs. The experiment was conducted in triplicates. The Data are displayed as mean ± SEM. Statistical analysis was performed by one-way ANOVA analysis and a post hoc Tukey test. The significance of the statistics is given in comparison to native + LPS (### p ≤ 0.001) and shCTR + LPS (** p ≤ 0.01 and *** p ≤ 0.001). All other combinations were not significant in the one-way ANOVA and the following Tukey test

Article Snippet: The gene-specific assays as well as the Fast Advanced Mastermix were purchased from Life Technologies: Rps29 Mm02342448_gH (housekeeping gene), Tak1 (Map3k7) Mm00554514_m1.

Techniques: Genetically Modified, Comparison

Journal: Molecular and Cellular Biochemistry

Article Title: The inflammatory signalling mediator TAK1 mediates lymphocyte recruitment to lipopolysaccharide-activated murine mesenchymal stem cells through interleukin-6

doi: 10.1007/s11010-021-04180-8

Figure Lengend Snippet: Analysis of LPS‐secreted factors in murine MSCs in a TAK1‐dependent mode. (A) LPS‐stimulated cytokine secretion in native mMSCs. mMSCs were cultivated in reduced serum levels (5%) for 72 h in the presence or absence of LPS (1 μg/ml). LPS induces/upregulates 5 factors (red arrows) out of 10 secreted cytokines in native mMSCs. (B) Expression levels were quantified as pixel density as determined by image analysis. (C) LPS‐stimulated cytokine secretion in mMSCs infected with lentiviruses encoding small hairpin control RNA (shCTR) or shRNA specific for TAK1 (shTAK1). IL‐6 is the only secreted cytokine detected which is substantially regulated by TAK1 signalling. (D) Expression levels were quantified as pixel density as determined by image analysis. TAK1 downregulation leads to a reduction of the IL‐6 secretion level to 46.5% (red arrows). (E) Native mMSCs were exposed to 5 µM TAK1 inhibitor 5Z-7-oxozeanol or solvent one hour prior to stimulation and then stimulated with LPS (1 µg/ml) for the time periods indicated. MW marker 100 bp (range 100—1000 bp)

Article Snippet: The gene-specific assays as well as the Fast Advanced Mastermix were purchased from Life Technologies: Rps29 Mm02342448_gH (housekeeping gene), Tak1 (Map3k7) Mm00554514_m1.

Techniques: Expressing, Infection, Control, shRNA, Solvent, Marker