Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: TNFSF9 induces M2 polarization of macrophages and promotes the migration of pancreatic cancer cells. ( A ) U937 cells were successfully induced into macrophages. ( B , C ) The effect of wild-type pancreatic cancer cells on the polarization of U937-derived macrophages. ( D , E ) TNFSF9 knockdown pancreatic cancer cells inhibit the expression of M2 markers (IL-10 and TGF-β) mRNA levels in U937 macrophages. At the same time, it promotes the expression of M1 marker (IL-8 and TNF-α) mRNA level and inhibits the expression of IL-1β mRNA level. ( F , G ) Co-culture of U937-derived macrophages with TNFSF9 knockdown pancreatic cancer cells inhibited the migration of BXPC-3 and PANC-1 cells. ( H , I ) The migration of BXPC-3 and PANC-1 cells increased after adding recombinant human protein TGF-β. ( J – L ) Western blots were used to analyze the expression of inflammatory cytokines IL-8, IL-6, TNF-α and IL-1β on U937-derived macrophages after co-culture with pancreatic cancer cells knocked down by TNFSF9. Compared with the sh-NC group, the expression of IL-8, IL-6 and TNF-α in the sh-TNFSF9-1 and sh-TNFSF9-2 groups were significantly increased, and the expression of IL-1β was significantly reduced. sh-1 is sh-TNFSF9-1. sh-2 is sh-TNFSF9-2. * P < 0.05, ** P < 0.01, *** P < 0.001 and # P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Migration, Derivative Assay, Expressing, Marker, Co-Culture Assay, Recombinant, Western Blot

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: TNFSF9 activates Wnt signal to promote M2 polarization of macrophages. ( A ) The mRNA expression levels of IL-10 and TGF-β in TNFSF9 knockdown pancreatic cancer cells were decreased, and the mRNA expression levels of IL-4 were increased. ( B ) After adding Wnt agonist, the mRNA expression level of IL-10 and TGF-β increased. ( C ) After adding Wnt agonist, pancreatic cancer cells that knock down TNFSF9 reduce the mRNA levels of M1 markers (IL-1β, IL-8 and TNF-α) in macrophages. ( D ) After adding Wnt agonist, pancreatic cancer cells that knock down TNFSF9 increase the mRNA levels of M2 markers (IL-4, IL-10 and TGF-β) in macrophages. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Cell Type Dependent Suppression of Inflammatory Mediators by Myocardin Related Transcription Factors

doi: 10.3389/fphys.2021.732564

Figure Lengend Snippet: The anti-inflammatory impact of MRTF-A is maintained in pro-inflammatory conditions. (A) Human coronary artery smooth muscle cells in culture were transduced with Null and MRTF-A viruses in control conditions (open bars), or with simultaneous treatment with lipopolysaccharide (LPS, 500 ng/ml), to mimic a pro-inflammatory environment (gray bars). After harvesting the cells, RNA was isolated, and inflammatory transcripts were assayed using RT-qPCR. The fold repression by MRTF-A is indicated below the asterisks that indicate significance, showing that MRTF-A was similarly effective in the absence and presence of LPS. CCL2 for example was reduced eightfold in control conditions and sevenfold in LPS treated cells, and an independent analysis indicated that this difference was not significant. To support MRTF-dependent reductions of inflammatory mediators at the protein level, MCP-1 ( CCL2 , (B,D) ) and IL-8 ( CXCL8 , (C,E) ) were examined using western blotting (B,C) and ELISAs (D,E) . All assays indicted significant MRTF-A-dependent reductions at the protein level, both in the absence and presence of LPS. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The membrane strips were then incubated with monoclonal primary antibodies as follows: MCP-1 (CCL2, 1 μg/ml, Abcam, ab9669), IL8 (CXCL8, 1:500, Cell Signaling, #94407), RelA (NFκB/p65, 1:1000, Cell Signaling, #8242), RelB (1:1000, Cell Signaling, #10544), NFκB1 (p105/p50, 1:1000, Cell Signaling, #13586), NFκB2 (p100/p52, 1:1000, Cell Signaling, #4882), HSP90 (1:1000, BD Biosciences, #610418), and GAPDH (1:3000, Merck Millipore, #MAB374) for 96 h at 4°C.

Techniques: Transduction, Control, Isolation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Lipid Emulsion Improves Functional Recovery in an Animal Model of Stroke

doi: 10.3390/ijms21197373

Figure Lengend Snippet: Effects of LE or vehicle on protein expressions after MCAO and reperfusion injury. ( a ) Representative Western blots indicating the expression of specific proteins in the penumbra region of the left hemisphere; ( b – d ) Expression and phosphorylation of Akt in the experimental groups. pAkt levels in MCAO+Veh group decreased significantly compared to the Sham+Veh group. pAkt was significantly increased in the MCAO+LE 20% group compared to the MCAO+Veh group; ( e – g ) Expression and phosphorylation levels of GSK-3β (pGSK-3β) in the experimental groups. pGSK-3β and GSK-3β levels in the MCAO+Veh group was significantly lower than the Sham+Veh group. The MCAO+LE 20% had significantly increased pGSK-3β and GSK-3β levels compared to the MCAO+Veh group; ( h – m ) Wnt signal-related protein expressions of experimental groups. Decreased levels of Wnt1, Wnt3, PORCN and β-catenin were observed in the MCAO+Veh group compared to the Sham+Veh group. The MCAO+LE 20% group had significantly increased protein levels of Wnt1, PORCN and β-catenin compared to MCAO+Veh group. Increased levels of pβ-catenin and tankyrase 1 were observed in the MCAO+Veh group compared to the Sham+Veh group. The MCAO+LE 20% group had significantly decreased levels of pβ-catenin and tankyrase 1 compared to the MCAO+Veh group; ( n – q ) Inflammatory protein expressions of experimental groups. Significantly increased expressions of inflammatory markers were observed in MCAO-injured groups compared to the Sham+Veh group. The MCAO+LE 20% group had significantly decreased inflammatory protein expression levels compared to the MCAO+Veh group. Data are presented as mean ± standard error of the mean (SEM); n = 8 for each group; * p < 0.05, ** p < 0.01 vs. Sham+Veh, # p < 0.05, ### p < 0.001 vs. MCAO+Veh, one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The following antibodies were used—anti-Wnt1 (ab15251, 1:1000, Abcam, Cambridge, UK), anti-Wnt3 (ab32249, Abcam), anti-PORCN (ab105543, Abcam), anti-β-catenin (#9562, 1:3000, Cell Signaling Technology, Beverly, MA, USA), anti-Akt (#4691, 1:3000, Cell Signaling Technology), anti-Phospho-Akt (#9271, 1:1000, Cell Signaling Technology), anti-GSK3-β (#9315, 1:3000, Cell Signaling Technology), anti-Phospho-GSK3-β (#9336, 1:1000, Cell Signaling Technology), anti-pβ-catenin (#9561, 1:1000, Cell Signaling Technology), anti-tankyrase 1 (MBS8531631, 1:1000, MyBioSource, San Diego, CA, USA), anti-IL-1β (AB1832P, 1:1000, Sigma Aldrich), anti-IL-6 (ARC0062, 1:500, Invitrogen, Carlsbad, CA, USA), anti-IL-8 (MBS9385550, 1:500, MyBioSource), anti-TNF-α (AAR33, 1:1000, Bio-Rad, Hercules, CA, USA) and anti-β-actin (#4967, 1:10,000, Cell Signaling Technology).

Techniques: Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Lipid Emulsion Improves Functional Recovery in an Animal Model of Stroke

doi: 10.3390/ijms21197373

Figure Lengend Snippet: Effects of LE or vehicle on protein expression on the MCAO and reperfusion injury after the administration of DMSO or XAV939. ( a ) Representative Western blots of proteins in the penumbra region of the left hemisphere; ( b – d ) Levels of Akt and pAkt in the experimental groups. The DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group decreased significantly in pAkt level compared to DMSO+Sham+Veh group. pAkt was significantly increased in the DMSO+MCAO+LE 20% group compared to the DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group; ( e – g ) Levels of GSK-3β and pGSK-3β in the experimental groups. GSK-3β and pGSK-3β levels of DMSO+MCAO+Veh group and XAV939+MCAO+LE 20% group were significantly lower than the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% had significantly increased pGSK-3β and GSK-3β levels compared to the DMSO+MCAO+Veh and XAV939+MCAO+LE 20% groups; ( h – m ) Wnt signal-related protein expressions of experimental groups. Significantly decreased levels of Wnt1, Wnt3, PORCN and β-catenin were observed in the DMSO+MCAO+Veh group compared to the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% group had significantly increased protein levels of Wnt1 and PORCN compared to the DMSO+MCAO+Veh group. The XAV939+MCAO+LE 20% group had decreased expression levels of Wnt1 and PORCN compared to the DMSO+MCAO+LE 20% group. The β–catenin expression level in the DMSO+MCAO+LE 20% and XAV939+MCAO+LE 20% groups did not differ significantly. There was a significant increase in pβ–catenin and tankyrase 1 in the DMSO+MCAO+Veh compared to the DMSO+Sham+Veh group. Pβ–catenin and tankyrase1 was significantly decreased in the DMSO+MCAO+LE 20% compared to the DMSO+MCAO+Veh group. The XAV939+MCAO+LE 20% group had significantly increased pβ–catenin and tankyrase 1 expression levels compared to the DMSO+MCAO+LE 20% group; ( n – q ) Inflammatory protein expressions in the experimental groups. Significantly increased expressions of inflammatory markers were observed in the MCAO-injured groups compared to the DMSO+Sham+Veh group. The DMSO+MCAO+LE 20% group had significantly decreased inflammatory protein expression levels compared to the DMSO+MCAO+Veh group. Significant decrease in inflammatory protein expressions were observed in the XAV939+MCAO+LE 20% group compared to the DMSO+Sham+Veh group. TNF-α expression level was increased significantly in the XAV939+MCAO+LE 20% group compared to the DMSO+MCAO+LE 20% group. Data are presented as mean ± standard error of the mean (SEM); n = 8 for each group; * p < 0.05, ** p < 0.01 vs. DMSO+Sham+Veh, # p < 0.05, ### p < 0.001 vs. DMSO+MCAO+Veh, † p < 0.05, ††† p < 0.001 vs. DMSO+MCAO+LE 20%, one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The following antibodies were used—anti-Wnt1 (ab15251, 1:1000, Abcam, Cambridge, UK), anti-Wnt3 (ab32249, Abcam), anti-PORCN (ab105543, Abcam), anti-β-catenin (#9562, 1:3000, Cell Signaling Technology, Beverly, MA, USA), anti-Akt (#4691, 1:3000, Cell Signaling Technology), anti-Phospho-Akt (#9271, 1:1000, Cell Signaling Technology), anti-GSK3-β (#9315, 1:3000, Cell Signaling Technology), anti-Phospho-GSK3-β (#9336, 1:1000, Cell Signaling Technology), anti-pβ-catenin (#9561, 1:1000, Cell Signaling Technology), anti-tankyrase 1 (MBS8531631, 1:1000, MyBioSource, San Diego, CA, USA), anti-IL-1β (AB1832P, 1:1000, Sigma Aldrich), anti-IL-6 (ARC0062, 1:500, Invitrogen, Carlsbad, CA, USA), anti-IL-8 (MBS9385550, 1:500, MyBioSource), anti-TNF-α (AAR33, 1:1000, Bio-Rad, Hercules, CA, USA) and anti-β-actin (#4967, 1:10,000, Cell Signaling Technology).

Techniques: Expressing, Western Blot

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: ( A,B ) MTT and flow cytometry assays were used to determine the viability and apoptosis of LPS-induced HUVECs, respectively. ( C,D ) The levels of apoptosis-related proteins Bcl-2, Bax, cleaved-caspase3 and inflammation-related cytokines TNFα, IL-6, IL-8 and IL-1β in LPS-treated HUVECs were detected by western blot. ( E–G ) The productions of ROS, SOD and MDA were measured by DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: HUVECs were treated with LPS for 48 h, and cells in control group were not given any treatment and after LPS-treated HUVECs were transfected with si-HULC or si-NC. ( A ) HULC expression was measured by qRT-PCR. ( B,C ) MTT and Flow cytometry assays were performed to detect cell viability and apoptosis, respectively. ( D,E ) The levels of Bcl-2, Bax, cleaved-caspase3, TNFα, IL-6, IL-8 and IL-1β were determined by western blot. ( F–H ) ROS, SOD and MDA productions were examined by DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: HUVECs were treated with LPS for 48 h, and cells in control group were not given any treatment, and then LPS-treated HUVECs were transfected with si-NC, si-HULC, si-HULC + anti-miR-NC or si-HULC + anti-miR-204-5p. ( A ) MiR-204-5p expression was detected by qRT-PCR. ( B,C ) The cell viability and apoptosis were evaluated by using MTT and Flow cytometry assays, respectively. ( D,E ) Western blot assay was performed to examine the protein levels of Bcl-2, Bax, cleaved-caspase3, TNFα, IL-6, IL-8 and IL-1β. ( F – H ) ROS, SOD and MDA productions were determined by using DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: After LPS-treated HUVECs were transfected with miR-NC, miR-204-5p, miR-204-5p + vector or miR-204-5p + TRPM7, and cells in control group were not given any treatment. ( A,B ) TRPM7 mRNA and protein levels were measured by qRT-PCR and western blot, respectively. ( C,D ) The viability and apoptosis of transfected HUVECs were assessed by MTT and Flow cytometry assays, respectively. ( E,F ) The protein levels of Bcl-2, Bax, cleaved-caspase3, TNFα, IL-6, IL-8 and IL-1β were detected using western blot assay. ( G–I ) The productions of ROS, SOD and MDA were assessed by DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: ( A,B ) MTT and flow cytometry assays were used to determine the viability and apoptosis of LPS-induced HUVECs, respectively. ( C,D ) The levels of apoptosis-related proteins Bcl-2, Bax, cleaved-caspase3 and inflammation-related cytokines TNFα, IL-6, IL-8 and IL-1β in LPS-treated HUVECs were detected by western blot. ( E–G ) The productions of ROS, SOD and MDA were measured by DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: HUVECs were treated with LPS for 48 h, and cells in control group were not given any treatment and after LPS-treated HUVECs were transfected with si-HULC or si-NC. ( A ) HULC expression was measured by qRT-PCR. ( B,C ) MTT and Flow cytometry assays were performed to detect cell viability and apoptosis, respectively. ( D,E ) The levels of Bcl-2, Bax, cleaved-caspase3, TNFα, IL-6, IL-8 and IL-1β were determined by western blot. ( F–H ) ROS, SOD and MDA productions were examined by DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: HUVECs were treated with LPS for 48 h, and cells in control group were not given any treatment, and then LPS-treated HUVECs were transfected with si-NC, si-HULC, si-HULC + anti-miR-NC or si-HULC + anti-miR-204-5p. ( A ) MiR-204-5p expression was detected by qRT-PCR. ( B,C ) The cell viability and apoptosis were evaluated by using MTT and Flow cytometry assays, respectively. ( D,E ) Western blot assay was performed to examine the protein levels of Bcl-2, Bax, cleaved-caspase3, TNFα, IL-6, IL-8 and IL-1β. ( F – H ) ROS, SOD and MDA productions were determined by using DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: Bioscience Reports

Article Title: LPS promotes the progression of sepsis by activation of lncRNA HULC/miR-204-5p/TRPM7 network in HUVECs

doi: 10.1042/BSR20200740

Figure Lengend Snippet: After LPS-treated HUVECs were transfected with miR-NC, miR-204-5p, miR-204-5p + vector or miR-204-5p + TRPM7, and cells in control group were not given any treatment. ( A,B ) TRPM7 mRNA and protein levels were measured by qRT-PCR and western blot, respectively. ( C,D ) The viability and apoptosis of transfected HUVECs were assessed by MTT and Flow cytometry assays, respectively. ( E,F ) The protein levels of Bcl-2, Bax, cleaved-caspase3, TNFα, IL-6, IL-8 and IL-1β were detected using western blot assay. ( G–I ) The productions of ROS, SOD and MDA were assessed by DCFH-DA method and a commercial kit. * P <0.05.

Article Snippet: The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Flow Cytometry