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Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, <t>p-NF-κBp65/NF-κBp65,</t> and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.
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Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, <t>p-NF-κBp65/NF-κBp65,</t> and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.
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Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, <t>p-NF-κBp65/NF-κBp65,</t> and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.
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Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, <t>p-NF-κBp65/NF-κBp65,</t> and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.
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Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, <t>p-NF-κBp65/NF-κBp65,</t> and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.
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Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, <t>p-NF-κBp65/NF-κBp65,</t> and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.
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Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.

Journal: Cells

Article Title: Effects of Adipose-Derived Mesenchymal Stem Cell-Secretome on Pyroptosis of Laparoscopic Hepatic Ischemia Reperfusion Injury in a Porcine Model.

doi: 10.3390/cells14100722

Figure Lengend Snippet: Figure 6. ADSC-secretome inhibited the HMGB1/TLR4/NF-κB signaling pathway during HIRI. (a) Representative Western blot analysis of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB. (b–e) The levels of HMGB1, TLR4, p-NF-κBp65/NF-κBp65, and p-IκB proteins were evaluated. The protein levels of HMGB1, p-NF-κBp65, and NF-κBp65 were adjusted against the respective levels of β-actin. For TLR4 and p-IκB, their protein expression levels were normalized to α-tubulin levels. (f–h) The gene expression analysis for HMGB1, TLR4, and NF-κBp65 was conducted. Three replicates were performed for each sample. The relative mRNA levels were standardized with reference to β-actin. The significance of results is indicated as ▲p < 0.05, ▲▲p < 0.01, when compared to the IRI group; * p < 0.01 and ** p < 0.01, relative to the DMEM group; # p < 0.05 and ## p < 0.01, in comparison to the ADSCs group.

Article Snippet: Following this, the membranes were incubated overnight with primary antibodies specific to β-actin (1:1000, Cell Signaling Technology, MA, USA), α-tubulin (1:10,000, Proteintech, IL, USA), IL-1β (1:500, ABmart, Shanghai, China), IL-18, caspase1, NLRP3, HMGB1, TLR4 (1:1000, WanLei Biologicals, Shenyang, China), apoptosis-associated speck-like protein containing a CARD (ASC), GSDMD-N (1:1000, Affinity Biosciences, FLA, USA), NF-κBp65 (1:2000, Proteintech, IL, USA), p-IκB (1:1000, Immunoway, TX, USA), p-NF-κBp65 (1:500, Santa Cruz Biotechnology, CA, USA), and IκB (1:1000, Immunoway, TX, USA).

Techniques: Western Blot, Expressing, Gene Expression, Comparison