anti-apaf1 antibody  (Advisains)

Journal: The Journal of Clinical Investigation

Article Title: Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation

doi: 10.1172/JCI161935

Figure Lengend Snippet: ( A ) Identification of HERC6 as a potential NLRP3 interactor in LPS-stimulated mouse PMs by mass spectrometry. ( B and C ) Immunoblot analysis of HERC6 and ISG15 expression in LPS-stimulated ( B ) or mIL-1β–stimulated ( C ) mouse PMs. ( D and E ) RT-PCR analysis of Herc6 ( D ) or Isg15 ( E ) mRNA in LPS-stimulated or mIL-1β–stimulated mouse PMs. All data are presented as the mean ± SD. ( F ) Co-IP analysis of the association between mHERC6 and mNLRP3 in HEK293T cells transfected with the indicated plasmids. ( G ) Co-IP analysis of the association between hNLRP3 and hHERC5 or hHERC6 in HEK293T cells transfected with the indicated plasmids. ( H ) Co-IP analysis of the association between HERC5 and NLRP3, ASC, or CASP1 in HEK293T cells transfected with the indicated plasmids. ( I ) Myc-NLRP3 and Flag-HERC5 were obtained by in vitro transcription and translation. The interaction between NLRP3 and HERC5 was assayed by mixing recombinant Myc-NLRP3 and Flag-HERC5, followed by co-IP with Myc antibody and immunoblot analysis with Flag or Myc antibody. ( J ) Co-IP analysis of the endogenous association between HERC6 and NLRP3, ASC, CASP1, or NEK7 in LPS-stimulated or LPS-primed and ATP-activated mouse PMs. ( K ) Co-IP analysis of the endogenous association between HERC5 and NLRP3, ASC, CASP1, or NEK7 in LPS-stimulated or LPS-primed and ATP-activated THP-1 cells. ( L ) Co-IP analysis of the association between NLRP3 and HERC5, ARIH1, or TRIM25 in HEK293T cells transfected with the indicated plasmids. Similar results were obtained from 3 independent experiments.

Article Snippet: ATP (A1852), Nig sodium salt (N7143), Z-Leu-Leu-Leu-al (MG132, C2211), LPS ( E . coli , O111:B4, L4130), anti-Myc (M4439, 1:5,000), anti-HA (H3663, 1:1,000), and anti-Flag M2 (F1804, 1:1,000) were obtained from MilliporeSigma; CHX (A8244) was from APExBIO Technology; poly(dA:dT) (tlrl-patn), flagellin (tlrl-epstfla), and MSU Crystals (tlrl-msu-25) were from Invivogen; recombinant human ISG15 protein (UL-601), recombinant human ISG15 E1/UBE1L protein (E-309), recombinant human UBCH8/UBE2L6 protein (E2-644-100), and 10× ubiquitin conjugation reaction buffer were from R&D Systems; mouse IFNB1/IFN-beta/Interferon beta Protein (50708-MCCH) was from Sino Biological; recombinant murine IL-1β (211-11B) was from PeproTech China; anti-mouse IgG (7076, 1:5,000), anti–p-IκBα (9246, 1:1,000), anti-IκBα (4814, 1:1,000), anti-AIM2 (13095S, 1:1,000), and anti-ISG15 (2743, 1:1,000) were from Cell Signaling Technology; anti–caspase-1 p20 (AG-20B-0042, 1:1,000), anti-NLRP3 (AG-20B-0014, 1:1,000), and anti-ASC (AG-25B-0006, 1:1,000) were from AdipoGen; anti-Ub (sc-8017, 1:1,000) and protein G agarose (sc-2002) used for IP were from Santa Cruz Biotechnology Inc.; anti-NLRP3 (ab263899, 1:1,000), anti-NEK7 (ab133514, 1:1,000), anti–caspase-1 (ab179515, 1:1,000), anti-HERC6 (ab22553, 1:1,000), and anti–IL-1β (ab234437, 1:1,000) were from Abcam; anti-NLRP3 (19771-1-AP, 1:1,000), anti-His (66005-1-Ig, 1:1,000), anti-HERC5 (22692-1-AP, 1:1,000), HRP-conjugated goat anti-rabbit IgG (H+L) (SA00001-2, 1:5,000), anti–β-actin (66009-1-Ig, 1:2,000), HRP-conjugated IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (SA00001-7L, 1:5,000), and HRP-conjugated Recombinant Rabbit Anti-Mouse IgG, Kappa Light Chain (SA00001-19, 1:5,000) were from Proteintech; goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody and alum (catalog 77161) were from Thermo Fisher Scientific; anti-Myc (N7143, 1:1,000) and purified recombinant NLRP3 (WX030FB0) were from Origene.

Techniques: Mass Spectrometry, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Transfection, In Vitro, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation

doi: 10.1172/JCI161935

Figure Lengend Snippet: ( A and B ) ELISA analysis of IL-1β, TNF-α, and IL-6 in supernatants of PMs from Herc6 +/+ or Herc6 –/– mice following priming with LPS for 7 hours and stimulation with ATP or Nig for 1 hour ( A : 2-tailed t test, Herc6 +/+ vs. Herc6 –/– , * P = 0.012074, *** P = 0.000040). ( C ) ELISA analysis of IL-1β in supernatants of PMs from Herc6 +/+ or Herc6 –/– mice following LPS priming for 7 hours and stimulation with poly(dA:dT) or flagellin for 1 hour. ( D ) Immunoblot analysis of supernatants (SN) and cell lysates (CL) of PMs from Herc6 +/+ or Herc6 –/– mice, following LPS priming and subsequent ATP stimulation for 1 hour. ( E ) ELISA analysis of IL-1β in supernatants from PMs transfected with control (Ctrl) siRNA or Herc6 siRNA for 48 hours, followed by priming with LPS for 7 hours and stimulation with ATP, Nig, or poly(dA:dT) for 1 hour (2-tailed t test, Ctrl siRNA vs. Herc6 siRNA, ** P = 0.009794, * P = 0.018808). ( F ) ELISA analysis of TNF-α and IL-6 in supernatants from PMs transfected with Ctrl or Herc6 siRNA for 48 hours, followed by priming with LPS for 7 hours and stimulation with ATP or Nig for 1 hour. ( G ) Immunoblot analysis of SN and CL of mouse PMs transfected with Ctrl siRNA or Herc6 siRNA for 48 hours, followed by LPS priming and ATP stimulation for 1 hour. ( H and I ) Immunoblot analysis of p-IκBα in LPS-stimulated PMs from Herc6 +/+ or Herc6 –/– mice ( H ), or PMs transfected with Ctrl or Herc6 siRNA ( I ). ( J and K ) RT-PCR analysis of Nlrp3 or Il1b , Tnfa, and Il6 mRNA in LPS-stimulated PMs from Herc6 +/+ or Herc6 –/– mice. All data are presented as the mean ± SD in A – C , E , F , J , and K . Similar results were obtained from 3 independent experiments.

Article Snippet: ATP (A1852), Nig sodium salt (N7143), Z-Leu-Leu-Leu-al (MG132, C2211), LPS ( E . coli , O111:B4, L4130), anti-Myc (M4439, 1:5,000), anti-HA (H3663, 1:1,000), and anti-Flag M2 (F1804, 1:1,000) were obtained from MilliporeSigma; CHX (A8244) was from APExBIO Technology; poly(dA:dT) (tlrl-patn), flagellin (tlrl-epstfla), and MSU Crystals (tlrl-msu-25) were from Invivogen; recombinant human ISG15 protein (UL-601), recombinant human ISG15 E1/UBE1L protein (E-309), recombinant human UBCH8/UBE2L6 protein (E2-644-100), and 10× ubiquitin conjugation reaction buffer were from R&D Systems; mouse IFNB1/IFN-beta/Interferon beta Protein (50708-MCCH) was from Sino Biological; recombinant murine IL-1β (211-11B) was from PeproTech China; anti-mouse IgG (7076, 1:5,000), anti–p-IκBα (9246, 1:1,000), anti-IκBα (4814, 1:1,000), anti-AIM2 (13095S, 1:1,000), and anti-ISG15 (2743, 1:1,000) were from Cell Signaling Technology; anti–caspase-1 p20 (AG-20B-0042, 1:1,000), anti-NLRP3 (AG-20B-0014, 1:1,000), and anti-ASC (AG-25B-0006, 1:1,000) were from AdipoGen; anti-Ub (sc-8017, 1:1,000) and protein G agarose (sc-2002) used for IP were from Santa Cruz Biotechnology Inc.; anti-NLRP3 (ab263899, 1:1,000), anti-NEK7 (ab133514, 1:1,000), anti–caspase-1 (ab179515, 1:1,000), anti-HERC6 (ab22553, 1:1,000), and anti–IL-1β (ab234437, 1:1,000) were from Abcam; anti-NLRP3 (19771-1-AP, 1:1,000), anti-His (66005-1-Ig, 1:1,000), anti-HERC5 (22692-1-AP, 1:1,000), HRP-conjugated goat anti-rabbit IgG (H+L) (SA00001-2, 1:5,000), anti–β-actin (66009-1-Ig, 1:2,000), HRP-conjugated IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (SA00001-7L, 1:5,000), and HRP-conjugated Recombinant Rabbit Anti-Mouse IgG, Kappa Light Chain (SA00001-19, 1:5,000) were from Proteintech; goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody and alum (catalog 77161) were from Thermo Fisher Scientific; anti-Myc (N7143, 1:1,000) and purified recombinant NLRP3 (WX030FB0) were from Origene.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation

doi: 10.1172/JCI161935

Figure Lengend Snippet: ( A ) Immunoblot analysis of lysates of mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours, following LPS stimulation. ( B ) Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3 and increasing amounts of HA-ISG15 plasmid. ( C ) Immunoblot analysis of lysates of PMs from Ifnar +/+ or Ifnar –/– mice, following LPS stimulation for 8 hours. ( D ) RT-PCR analysis of Nlrp3 mRNA in LPS-stimulated mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours. ( E ) ELISA analysis of IL-1β in supernatants from mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours, followed by priming with LPS for 7 hours and subsequent stimulation with ATP, Nig, or poly(dA:dT) for 1 hour (2-tailed t test, Ctrl siRNA vs. Isg15 siRNA, * P = 0.022242, ** P = 0.001884). ( F ) Immunoblot analysis of SN and CL from mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours, followed by LPS priming and subsequent ATP stimulation for 1 hour. All data are presented as mean ± SD in D and E . Similar results were obtained from 3 independent experiments.

Article Snippet: ATP (A1852), Nig sodium salt (N7143), Z-Leu-Leu-Leu-al (MG132, C2211), LPS ( E . coli , O111:B4, L4130), anti-Myc (M4439, 1:5,000), anti-HA (H3663, 1:1,000), and anti-Flag M2 (F1804, 1:1,000) were obtained from MilliporeSigma; CHX (A8244) was from APExBIO Technology; poly(dA:dT) (tlrl-patn), flagellin (tlrl-epstfla), and MSU Crystals (tlrl-msu-25) were from Invivogen; recombinant human ISG15 protein (UL-601), recombinant human ISG15 E1/UBE1L protein (E-309), recombinant human UBCH8/UBE2L6 protein (E2-644-100), and 10× ubiquitin conjugation reaction buffer were from R&D Systems; mouse IFNB1/IFN-beta/Interferon beta Protein (50708-MCCH) was from Sino Biological; recombinant murine IL-1β (211-11B) was from PeproTech China; anti-mouse IgG (7076, 1:5,000), anti–p-IκBα (9246, 1:1,000), anti-IκBα (4814, 1:1,000), anti-AIM2 (13095S, 1:1,000), and anti-ISG15 (2743, 1:1,000) were from Cell Signaling Technology; anti–caspase-1 p20 (AG-20B-0042, 1:1,000), anti-NLRP3 (AG-20B-0014, 1:1,000), and anti-ASC (AG-25B-0006, 1:1,000) were from AdipoGen; anti-Ub (sc-8017, 1:1,000) and protein G agarose (sc-2002) used for IP were from Santa Cruz Biotechnology Inc.; anti-NLRP3 (ab263899, 1:1,000), anti-NEK7 (ab133514, 1:1,000), anti–caspase-1 (ab179515, 1:1,000), anti-HERC6 (ab22553, 1:1,000), and anti–IL-1β (ab234437, 1:1,000) were from Abcam; anti-NLRP3 (19771-1-AP, 1:1,000), anti-His (66005-1-Ig, 1:1,000), anti-HERC5 (22692-1-AP, 1:1,000), HRP-conjugated goat anti-rabbit IgG (H+L) (SA00001-2, 1:5,000), anti–β-actin (66009-1-Ig, 1:2,000), HRP-conjugated IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (SA00001-7L, 1:5,000), and HRP-conjugated Recombinant Rabbit Anti-Mouse IgG, Kappa Light Chain (SA00001-19, 1:5,000) were from Proteintech; goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody and alum (catalog 77161) were from Thermo Fisher Scientific; anti-Myc (N7143, 1:1,000) and purified recombinant NLRP3 (WX030FB0) were from Origene.

Techniques: Western Blot, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation

doi: 10.1172/JCI161935

Figure Lengend Snippet: ( A ) ELISA analysis of serum levels of IL-1β, TNF-α, and IL-6 of Herc6 +/+ or Herc6 –/– mice after i.p. LPS injection for 4 hours (PBS n = 3, LPS, n = 6 per group; 2-tailed t test, Herc6 +/+ vs. Herc6 –/– , ** P = 0.005057). ( B and C ) ELISA analysis of serum ( B ) or BALF ( C ) IL-1β levels of Herc6 +/+ or Herc6 –/– mice after i.p. LPS injection for 12 hours (PBS n = 3; LPS n = 5 per group; 2-tailed t test, Herc6 +/+ vs. Herc6 –/– , B : *** P = 1.36 × 10 –4 , C : *** P = 3.84 × 10 –5 ). ( D ) WBC count in BALF of IL-1β of Herc6 +/+ or Herc6 –/– mice after i.p. LPS injection for 12 hours (PBS n = 2, LPS, n = 6 per group; 2-tailed t test, Herc6 +/+ vs. Herc6 –/– , * P = 0.031741). ( E and F ) Immunoblot analysis of lysates from the lung, spleen, and liver of Herc6 +/+ or Herc6 –/– mice after i.p. LPS injection for 12 hours. NLRP3 expression was quantitated by measuring band intensities using ImageJ software. The values were normalized to actin (PBS n = 2, LPS n = 5 per group; 2-tailed t test, Herc6 +/+ vs. Herc6 –/– , lung: * P = 0.025147, spleen: ** P = 0.007801, liver: * P = 0.040026). ( G ) H&E staining of lung tissue sections from Herc6 +/+ or Herc6 –/– mice after i.p. LPS injection for 12 hours (PBS n = 2, LPS n = 4 per condition). Scale bars: 10 μm. All data are presented as mean ± SD in A – D and F . Similar results were obtained from 3 independent experiments.

Article Snippet: ATP (A1852), Nig sodium salt (N7143), Z-Leu-Leu-Leu-al (MG132, C2211), LPS ( E . coli , O111:B4, L4130), anti-Myc (M4439, 1:5,000), anti-HA (H3663, 1:1,000), and anti-Flag M2 (F1804, 1:1,000) were obtained from MilliporeSigma; CHX (A8244) was from APExBIO Technology; poly(dA:dT) (tlrl-patn), flagellin (tlrl-epstfla), and MSU Crystals (tlrl-msu-25) were from Invivogen; recombinant human ISG15 protein (UL-601), recombinant human ISG15 E1/UBE1L protein (E-309), recombinant human UBCH8/UBE2L6 protein (E2-644-100), and 10× ubiquitin conjugation reaction buffer were from R&D Systems; mouse IFNB1/IFN-beta/Interferon beta Protein (50708-MCCH) was from Sino Biological; recombinant murine IL-1β (211-11B) was from PeproTech China; anti-mouse IgG (7076, 1:5,000), anti–p-IκBα (9246, 1:1,000), anti-IκBα (4814, 1:1,000), anti-AIM2 (13095S, 1:1,000), and anti-ISG15 (2743, 1:1,000) were from Cell Signaling Technology; anti–caspase-1 p20 (AG-20B-0042, 1:1,000), anti-NLRP3 (AG-20B-0014, 1:1,000), and anti-ASC (AG-25B-0006, 1:1,000) were from AdipoGen; anti-Ub (sc-8017, 1:1,000) and protein G agarose (sc-2002) used for IP were from Santa Cruz Biotechnology Inc.; anti-NLRP3 (ab263899, 1:1,000), anti-NEK7 (ab133514, 1:1,000), anti–caspase-1 (ab179515, 1:1,000), anti-HERC6 (ab22553, 1:1,000), and anti–IL-1β (ab234437, 1:1,000) were from Abcam; anti-NLRP3 (19771-1-AP, 1:1,000), anti-His (66005-1-Ig, 1:1,000), anti-HERC5 (22692-1-AP, 1:1,000), HRP-conjugated goat anti-rabbit IgG (H+L) (SA00001-2, 1:5,000), anti–β-actin (66009-1-Ig, 1:2,000), HRP-conjugated IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (SA00001-7L, 1:5,000), and HRP-conjugated Recombinant Rabbit Anti-Mouse IgG, Kappa Light Chain (SA00001-19, 1:5,000) were from Proteintech; goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody and alum (catalog 77161) were from Thermo Fisher Scientific; anti-Myc (N7143, 1:1,000) and purified recombinant NLRP3 (WX030FB0) were from Origene.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Western Blot, Expressing, Software, Staining

Journal: The Journal of Clinical Investigation

Article Title: Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation

doi: 10.1172/JCI161935

Figure Lengend Snippet: ( A and B ) Immunoblot analysis of HERC6 and ISG15 expression in VSV-infected ( A ) or mIFN-β–stimulated ( B ) mouse PMs. ( C ) ELISA analysis of IL-1β in supernatants of PMs from Herc6 +/+ or Herc6 –/– mice after infection with VSV for 24 hours (2-tailed t test, Herc6 +/+ vs. Herc6 –/– , ** P = 0.003646). ( D ) Immunoblot analysis of PMs from Herc6 +/+ or Herc6 –/– mice after VSV infection. ( E ) ELISA analysis of TNF-α and IL-6 in supernatants of PMs from Herc6 +/+ or Herc6 –/– mice after VSV infection for 24 hours (2-tailed t test, Herc6 +/+ vs. Herc6 –/– , *** P = 0.000399, ** P = 0.004338). ( F ) ELISA analysis of IL-1β, TNF-α, and IL-6 in supernatants from mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours — following VSV infection for 12 or 24 hours (2-tailed t test, Ctrl vs. Isg15 siRNA, IL-1β: *** P = 0.000059, * P = 0.023051; TNF-α: * P = 0.011107 [12 h], * P = 0.026233 [24h]; IL-6: ** P = 0.001473 [12h], ** P = 0.001505 [24h] . ( G ) Immunoblot analysis of mouse PMs transfected with Ctrl or Isg15 siRNA for 48 hours, following VSV infection. ( H ) Immunoblot analysis of THP-1 cells transfected with Ctrl or HERC5 siRNA for 48 hours, following VSV infection. ( I – K ) ELISA analysis IL-1β in serum ( I ) and BALF ( J ), or TNF-α and IL-6 in serum ( K ) of Herc6 +/+ or Herc6 –/– mice after infection with VSV by i.p. injection for 12 hours (PBS n = 3, VSV n = 5 per group; 2-tailed t test, Herc6 +/+ vs. Herc6 –/– , I : *** P = 3.44 × 10 –5 , J : *** P = 0.000233, K : TNF-α *** P = 9.78 × 10 –7 , IL-6 *** P = 0.000241). All data are presented as mean ± SD in C , E , F , and I – K . Similar results were obtained from 3 independent experiments.

Article Snippet: ATP (A1852), Nig sodium salt (N7143), Z-Leu-Leu-Leu-al (MG132, C2211), LPS ( E . coli , O111:B4, L4130), anti-Myc (M4439, 1:5,000), anti-HA (H3663, 1:1,000), and anti-Flag M2 (F1804, 1:1,000) were obtained from MilliporeSigma; CHX (A8244) was from APExBIO Technology; poly(dA:dT) (tlrl-patn), flagellin (tlrl-epstfla), and MSU Crystals (tlrl-msu-25) were from Invivogen; recombinant human ISG15 protein (UL-601), recombinant human ISG15 E1/UBE1L protein (E-309), recombinant human UBCH8/UBE2L6 protein (E2-644-100), and 10× ubiquitin conjugation reaction buffer were from R&D Systems; mouse IFNB1/IFN-beta/Interferon beta Protein (50708-MCCH) was from Sino Biological; recombinant murine IL-1β (211-11B) was from PeproTech China; anti-mouse IgG (7076, 1:5,000), anti–p-IκBα (9246, 1:1,000), anti-IκBα (4814, 1:1,000), anti-AIM2 (13095S, 1:1,000), and anti-ISG15 (2743, 1:1,000) were from Cell Signaling Technology; anti–caspase-1 p20 (AG-20B-0042, 1:1,000), anti-NLRP3 (AG-20B-0014, 1:1,000), and anti-ASC (AG-25B-0006, 1:1,000) were from AdipoGen; anti-Ub (sc-8017, 1:1,000) and protein G agarose (sc-2002) used for IP were from Santa Cruz Biotechnology Inc.; anti-NLRP3 (ab263899, 1:1,000), anti-NEK7 (ab133514, 1:1,000), anti–caspase-1 (ab179515, 1:1,000), anti-HERC6 (ab22553, 1:1,000), and anti–IL-1β (ab234437, 1:1,000) were from Abcam; anti-NLRP3 (19771-1-AP, 1:1,000), anti-His (66005-1-Ig, 1:1,000), anti-HERC5 (22692-1-AP, 1:1,000), HRP-conjugated goat anti-rabbit IgG (H+L) (SA00001-2, 1:5,000), anti–β-actin (66009-1-Ig, 1:2,000), HRP-conjugated IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific (SA00001-7L, 1:5,000), and HRP-conjugated Recombinant Rabbit Anti-Mouse IgG, Kappa Light Chain (SA00001-19, 1:5,000) were from Proteintech; goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody and alum (catalog 77161) were from Thermo Fisher Scientific; anti-Myc (N7143, 1:1,000) and purified recombinant NLRP3 (WX030FB0) were from Origene.

Techniques: Western Blot, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Transfection, Injection

Journal: PLOS Biology

Article Title: GIRK2 potassium channels expressed by the AgRP neurons decrease adiposity and body weight in mice

doi: 10.1371/journal.pbio.3002252

Figure Lengend Snippet: (A) Bar graphs and dots summarize oxygen consumption (VO 2 ) (left, dark cycle: 3.23 ± 0.07 L/h/kg, n = 14, for GIRK2 WT and 2.98 ± 0.09 L/h/kg, n = 16, for GIRK2 AgRP-KO , df = 28, t = 2.088, p = 0.046; light cycle: 2.57 ± 0.06 L/h/kg, n = 14, for GIRK2 WT and 2.38 ± 0.07 L/h/kg, n = 16, for GIRK2 AgRP-KO , df = 28, t = 1.842, p = 0.076), carbon dioxide production (VCO 2 ) (middle, dark cycle: 2.99 ± 0.06 L/h/kg, n = 14, for GIRK2 WT and 2.66 ± 0.08 L/h/kg, n = 16, for GIRK2 AgRP-KO , df = 28, t = 3.198, p = 0.003; light cycle: 2.33 ± 0.04 L/h/kg, n = 14, for GIRK2 WT and 2.09 ± 0.07 L/h/kg, n = 16, for GIRK2 AgRP-KO , df = 28, t = 2.847, p = 0.008), and EE (right, dark cycle: 15.9 ± 0.3 kcal/h/kg, n = 14, for GIRK2 WT and 14.7 ± 0.4 kcal/h/kg, n = 16, for GIRK2 AgRP-KO , df = 28, t = 2.140, p = 0.041; light cycle: 12.6 ± 0.3 kcal/h/kg, n = 14, for GIRK2 WT and 11.7 ± 0.4 kcal/h/kg, n = 16, for GIRK2 AgRP-KO , df = 28, t = 1.972, p = 0.059) of GIRK2 WT ( n = 14) and GIRK2 AgRP-KO ( n = 16) mice measured by indirect calorimetry. (B) Images demonstrate HE (upper), oil red O (middle) staining, and UCP1 (lower) immunostaining results of BAT obtained from GIRK2 WT (left) and GIRK2 AgRP-KO (right) mice. Scale bar = 20 μm. (C, D) Images on the left demonstrate IHC of ChAT (red), Fos (green), and DAPI (blue) in upper (T1-T6) thoracic spinal cords of GIRK2 WT (C) and GIRK2 AgRP-KO (D) mice at a lower magnification. Scale bar = 100 μm. Areas of IML in the rectangles are shown on the right at a higher magnification. In merged images, gray arrowheads indicate Fos (−) and ChAT (+) neurons, and yellow arrowheads indicate Fos (+) and ChAT (+) neurons. Scale bar = 10 μm. (E) Bar graphs and dots summarize proportion of Fos-expressing ChAT neurons in IML of GIRK2 WT (52.4 ± 3.9%, n = 6) and GIRK2 AgRP-KO (32.4 ± 2.6%, n = 4) mice (df = 8, t = 3.79, p = 0.005). A total of 48 spinal cord slices from each mouse (levels T1-T6) were included for analyses. Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. * p < 0.05, ** p < 0.01. The numerical data for Fig 6A and 6E can be found in . BAT, brown adipose tissue; ChAT, choline acetyltransferase; EE, energy expenditure; GIRK, G protein-gated inwardly rectifying K + ; HE, hematoxylin and eosin; IHC, immunohistochemistry; IML, intermediolateral column.

Article Snippet: BAT was obtained from GIRK2 WT and GIRK2 AgRP-KO mice and was immediately submerged into formalin (HT501128, Sigma) for at least 24 h. Sections of BAT were treated with recombinant anti-UCP1 antibody (1:1,000, ab234430, Abcam), which was followed by incubation with horseradish peroxidase (HRP) secondary antibodies (Envision kit HRP, DAKO).

Techniques: Staining, Immunostaining, Expressing, Immunohistochemistry

Journal: Journal of Gastrointestinal Oncology

Article Title: Esophageal cancer stem cells reduce hypoxia-induced apoptosis by inhibiting the GRP78-perk-eIF2α-ATF4-CHOP pathway in vitro

doi: 10.21037/jgo-23-462

Figure Lengend Snippet: Effect of hypoxia on mitochondrial apoptosis pathway in EC cells (n=3). After unselected cells EC9706 and sorted CD44 + /CD24 − cells were cultured under normoxic and hypoxic conditions respectively, the protein levels of Bax, Apaf-1, caspase 9, and Bcl-2 in cells (A,B) and the expression of Cyt C protein in mitochondria and cytoplasm (C,D) were detected by WB. **, P<0.01; n.s., not significant. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, non-sorted; Cyt C, cytochrome C; EC, esophageal cancer; WB, western blot.

Article Snippet: High glucose-Dulbecco’s modified Eagle medium (H-DMEM; Gibco, Carlsbad, CA, USA); DMEM/F12 medium (Gibco); fetal bovine serum (FBS; Sijiqing Company, Hangzhou, China); 0.25% trypsin (T1300; Solarbio Co., Beijing, China); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; M8180; Solarbio Co.); JC-1 (MAK160; Sigma-Aldrich, Waltham, MA, USA); annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (CA1020; Solarbio Co.); cycle detection kit (CA1510; Solarbio Co.); Cyt C releasing apoptosis assay kit (Ab65311; Abcam, Cambridge, MA, USA); radioimmunoprecipitation assay (RIPA) lysis buffer (P0013C; Beyotime Co., Shanghai, China); horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (A0208; Beyotime Co.); real-time fluorescence quantitative polymerase chain reaction (qPCR) kit (RR820A; Bao Bioengineering Co., Ltd., Dalian, China); TRIeasy TM total RNA extraction reagent kit (9109; Bao Bioengineering Co., Ltd.); HyperScript III RT SuperMix for qPCR with genomic DNA (gDNA) Remover Kit [reverse transcription polymerase chain reaction (RT-PCR); RR047A; Bao Bioengineering Co., Ltd.]; rabbit anti-SOX2 polyclonal antibody (AB92494; Abcam); rabbit anti-OCT4 polyclonal antibody (AB200834; Abcam); rabbit anti-caspase 3 polyclonal antibody (AB13847; Abcam); rabbit anti-caspase 6 monoclonal antibody (AB185645; Abcam); rabbit anti-caspase 7 monoclonal antibody (AB255818; Abcam); phosphatidylethanolamine (PE)-conjugated anti-CD44 mouse monoclonal antibody (AB269300; Abcam); FITC-conjugated anti-CD24 mouse monoclonal antibody (AB30350; Abcam); HRP-labeled anti-rabbit secondary antibody (AB6721; Abcam); rabbit anti-caspase 9 monoclonal antibody (AB32539; Abcam); rabbit anti-Apaf-1 monoclonal antibody (AB234436; Abcam); rabbit anti-Bax monoclonal antibody (ab32503; Abcam); rabbit anti-Bcl-2 monoclonal antibody (ab32124; Abcam); rabbit anti-Cyt C monoclonal antibody (AB133504; Abcam); rabbit anti-VDAC1 monoclonal antibody (AB15895; Abcam); rabbit anti-Caspase 12 polyclonal antibody (ab62484; Abcam); rabbit anti-CHOP monoclonal antibody (ab11419; Abcam); rabbit anti-GRP78 monoclonal antibody (ab108615; Abcam); rabbit anti-JNK monoclonal antibody (ab199380; Abcam); rabbit anti-Ero1-Lα monoclonal antibody (ab177156; Abcam); rabbit anti-GADD34 monoclonal antibody (ab236516; Abcam); rabbit anti-TRIB3 monoclonal antibody (ab137526; Abcam); rabbit anti-ATF4 monoclonal antibody (ab270980; Abcam); rabbit anti-eIF2α monoclonal antibody (ab169528; Abcam); rabbit anti-p-eIF2α monoclonal antibody (YP0093; ImmunoWay Biotechnology, Plano, TX, USA); rabbit anti-PERK monoclonal antibody (ab229912; Abcam).

Techniques: Cell Culture, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Adipose Tissue-Derived Mesenchymal Stem Cell-Derived Exosomes Promote Wound Healing and Tissue Regeneration

doi: 10.3390/ijms241310434

Figure Lengend Snippet: Effects of ASC-EXOs on HDF proliferation, migration, and collagen synthesis. ( A ) HDFs were treated with positive control or ASC-EXOs in low (3 × 10 9 particles/mL) and high (1.5 × 10 10 particles/mL) concentrations for 24 h. Cell proliferation rate was determined by CCK-8 assays. ( B ) HDFs with 100% confluency in wells of a 96-well plate were scratched, and the migration results were photographed at 0 and 24 h. Purple indicates the cell area. ( C ) The scratch-wound assay recovery rate is presented as the percentage closure. ( D ) HDFs were treated with ASC-EXOs for 24 h, and mRNA expression levels of genes encoding collagen type I and type III, α-SMA, FGF2, and elastin (ELN) were analyzed by normalization to the GADPH levels. ( E ) Procollagen type 1 C-peptide concentration was determined after 24 h treatment of ASC-EXOs in HDFs. The data are shown as the mean ± standard deviation (SD) ( n = 3) with significance at * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For IHC, the dewaxed sections were washed with PBS, and endogenous peroxidase activity was quenched by immersion in 2% ( v / v ) hydrogen peroxide for 5 min. Antigen retrieval was performed by incubation with sodium citrate buffer for 30 min. After rinsing with PBS, the sections were blocked with 1.5% goat serum at room temperature for 30 min. Then, the sections were incubated with primary antibodies against collagen I (1:200; ab6308, Abcam, Cambridge, MA, USA) and collagen III (1:200; ab23445, Abcam, Cambridge, MA, USA).

Techniques: Migration, Positive Control, CCK-8 Assay, Scratch Wound Assay Assay, Expressing, Concentration Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Adipose Tissue-Derived Mesenchymal Stem Cell-Derived Exosomes Promote Wound Healing and Tissue Regeneration

doi: 10.3390/ijms241310434

Figure Lengend Snippet: Effects of ASC-EXOs on wound closure, re-epithelialization, and collagen synthesis in an extended porcine acute wound healing model. ( A ) Schematic diagram illustrating the skin wounds and the topical treatments in two SPF mini pigs. ( B ) Schedule of wound modeling, topical treatment, and tissue collection. ( C ) Comparison of wound closure rate between the combination (ASC-EXO + HA) treatment and HA alone. ( D ) Representative images of wound sites with each treatment on days 7, 11, 14, 21, 28, and 35. ( E ) Histological examination of skin tissue sections stained with H&E showing re-epithelialization (Re), inflammation (In), and granulation (Gn). Enlarged images of each section are shown in the right panel, corresponding to the boxed area in the left panel. Scale bars represent 1 mm. ( F ) Quantification of epithelialization score (0–3) indicating tissue repair. ( G ) Tissue sections were stained with Masson’s trichrome for visualization of collagen fibers and immunohistochemistry (IHC) for collagen type I and type III. Arrows indicate the edges of the remaining scar. Enlarged images of each section are shown in the right panel (scale bars: 200 μm), corresponding to the boxed area in the left panel (scale bars: 2000 μm). ( H ) Quantification of stained tissue area indicating collagen fiber, collagen type I, and type III. Data in the graphs are presented as the mean ± SD ( n = 6) with significance at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: For IHC, the dewaxed sections were washed with PBS, and endogenous peroxidase activity was quenched by immersion in 2% ( v / v ) hydrogen peroxide for 5 min. Antigen retrieval was performed by incubation with sodium citrate buffer for 30 min. After rinsing with PBS, the sections were blocked with 1.5% goat serum at room temperature for 30 min. Then, the sections were incubated with primary antibodies against collagen I (1:200; ab6308, Abcam, Cambridge, MA, USA) and collagen III (1:200; ab23445, Abcam, Cambridge, MA, USA).

Techniques: Staining, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Adipose Tissue-Derived Mesenchymal Stem Cell-Derived Exosomes Promote Wound Healing and Tissue Regeneration

doi: 10.3390/ijms241310434

Figure Lengend Snippet: Effects of ASC-EXOs on tissue regeneration in a mouse dermal filler model. ( A ) Schematic illustration showing the subcutaneous injection of HA or ASC-EXO + HA into the back of SKH1-hairless mice (6 weeks old, n = 4). ( B ) Representative images of explanted tissue graft 3 weeks after injection. ( C ) Histological evaluation of the grafts stained with H&E and quantification of infiltrated cells at 3 weeks post injection. Data are presented as the mean ± standard deviation (SD) with significance at ** p < 0.01. ( D ) Masson’s trichrome staining to visualize collagen fiber in blue and IHC staining for collagen type I ( E ) and type III ( F ). Data collected from three different areas of every slide are presented as the mean ± standard error of the means (SEM) with significance at * p < 0.05 and ** p < 0.01. Scale bars for ( C , D ), 50 µm. ( G ) Histological evaluation of the grafts for expression of α-SMA and vimentin. Representative IF images are shown, and the number of α-SMA-positive or/and vimentin-positive cells was quantified in three different areas of every tissue slide. Data are shown as the means ± SEM with significance at * p < 0.05 and *** p < 0.001. Scale bars represent 50 µm and 20 µm for the 20× and 40× magnification images, respectively.

Article Snippet: For IHC, the dewaxed sections were washed with PBS, and endogenous peroxidase activity was quenched by immersion in 2% ( v / v ) hydrogen peroxide for 5 min. Antigen retrieval was performed by incubation with sodium citrate buffer for 30 min. After rinsing with PBS, the sections were blocked with 1.5% goat serum at room temperature for 30 min. Then, the sections were incubated with primary antibodies against collagen I (1:200; ab6308, Abcam, Cambridge, MA, USA) and collagen III (1:200; ab23445, Abcam, Cambridge, MA, USA).

Techniques: Injection, Staining, Standard Deviation, Immunohistochemistry, Expressing

Journal: iScience

Article Title: Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization

doi: 10.1016/j.isci.2023.107130

Figure Lengend Snippet: LPS from the microbiota contributes to HCTZ-induced inflammation and insulin resistance (A and B) Cecal content (A) and serum (B) LPS levels in mice treated with HCTZ or vehicle control (n = 6–8 per group). ∗p < 0.05. Data are represented as mean ± SEM. (C) Correlation between serum LPS and HOMA-IR in the HFD-fed group (n = 5–8 per group). (D–F) Serum TNF-α (D), IL-1β (E), and IL-6 (F) in mice treated with HCTZ or vehicle control (n = 5–8 per group). ∗p < 0.05. Data are represented as mean ± SEM. (G) Serum LPS levels in recipient mice in the FMT experiment (n = 6–8 per group). ∗p < 0.05. Data are represented as mean ± SEM. (H–J) Serum TNF-α (H), IL-1β (I), and IL-6 (J) in recipient mice in the FMT experiment (n = 6–8 per group). ∗p < 0.05. Data are represented as mean ± SEM.

Article Snippet: Rabbit monoclonal anti- IL-1β , Abcam , Cat#ab234437;.

Techniques:

Journal: iScience

Article Title: Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization

doi: 10.1016/j.isci.2023.107130

Figure Lengend Snippet: HCTZ caused hepatic macrophage polarization and inflammation through a TLR4-dependent mechanism (A–E) Relative MyD88 (A) , NF-κB (B) , TNF-α (C) , IL-1β (D), and IL-6 (E) expression in the livers of mice treated with HCTZ or vehicle control (n = 5–8 per group). ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SEM. (F–K) Representative western blots (F) and quantification of MyD88 (G), NF-κB (H), TNF-α (I), IL-1β (J), and IL-6 (K) levels in the livers of mice treated with HCTZ or vehicle control (n = 3–4 per group). ∗p < 0.05. Data are represented as mean ± SEM. (L) Representative H&E staining in the livers of mice treated with HCTZ or vehicle control. (M and N) (M)Representative CD80 staining of hepatic F4/80 + macrophages in mice treated with HCTZ or vehicle control. (N) Flow cytometry analysis of CD80 + -F4/80 + macrophages (n = 3–4 per group). ∗p < 0.05. Data are represented as mean ± SEM. (O and P) (O)Representative CD206 staining of hepatic F4/80 + macrophages in mice treated with HCTZ or vehicle control. (P) Flow cytometry analysis of CD206 + -F4/80 + macrophages (n = 3–4 per group). ∗p < 0.05. Data are represented as mean ± SEM.

Article Snippet: Rabbit monoclonal anti- IL-1β , Abcam , Cat#ab234437;.

Techniques: Expressing, Western Blot, Staining, Flow Cytometry

Journal: iScience

Article Title: Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization

doi: 10.1016/j.isci.2023.107130

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti- IL-1β , Abcam , Cat#ab234437;.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization

doi: 10.1016/j.isci.2023.107130

Figure Lengend Snippet: LPS from the microbiota contributes to HCTZ-induced inflammation and insulin resistance (A and B) Cecal content (A) and serum (B) LPS levels in mice treated with HCTZ or vehicle control (n = 6–8 per group). ∗p < 0.05. Data are represented as mean ± SEM. (C) Correlation between serum LPS and HOMA-IR in the HFD-fed group (n = 5–8 per group). (D–F) Serum TNF-α (D), IL-1β (E), and IL-6 (F) in mice treated with HCTZ or vehicle control (n = 5–8 per group). ∗p < 0.05. Data are represented as mean ± SEM. (G) Serum LPS levels in recipient mice in the FMT experiment (n = 6–8 per group). ∗p < 0.05. Data are represented as mean ± SEM. (H–J) Serum TNF-α (H), IL-1β (I), and IL-6 (J) in recipient mice in the FMT experiment (n = 6–8 per group). ∗p < 0.05. Data are represented as mean ± SEM.

Article Snippet: After blocking with QuickBlock Western blocking buffer (Beyotime Biotechnology), the membrane was incubated with primary antibodies against MyD88 (Abcam, ab219413), NF-κB (Abcam, ab288751), TNF-α (Abcam, ab183896), IL-1β (Abcam, ab234437), and IL-6 (Abcam, ab259341) at 4°C overnight and probed with secondary antibodies conjugated with horseradish peroxidase for 1-2 h at room temperature.

Techniques:

Journal: iScience

Article Title: Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization

doi: 10.1016/j.isci.2023.107130

Figure Lengend Snippet: HCTZ caused hepatic macrophage polarization and inflammation through a TLR4-dependent mechanism (A–E) Relative MyD88 (A) , NF-κB (B) , TNF-α (C) , IL-1β (D), and IL-6 (E) expression in the livers of mice treated with HCTZ or vehicle control (n = 5–8 per group). ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SEM. (F–K) Representative western blots (F) and quantification of MyD88 (G), NF-κB (H), TNF-α (I), IL-1β (J), and IL-6 (K) levels in the livers of mice treated with HCTZ or vehicle control (n = 3–4 per group). ∗p < 0.05. Data are represented as mean ± SEM. (L) Representative H&E staining in the livers of mice treated with HCTZ or vehicle control. (M and N) (M)Representative CD80 staining of hepatic F4/80 + macrophages in mice treated with HCTZ or vehicle control. (N) Flow cytometry analysis of CD80 + -F4/80 + macrophages (n = 3–4 per group). ∗p < 0.05. Data are represented as mean ± SEM. (O and P) (O)Representative CD206 staining of hepatic F4/80 + macrophages in mice treated with HCTZ or vehicle control. (P) Flow cytometry analysis of CD206 + -F4/80 + macrophages (n = 3–4 per group). ∗p < 0.05. Data are represented as mean ± SEM.

Article Snippet: After blocking with QuickBlock Western blocking buffer (Beyotime Biotechnology), the membrane was incubated with primary antibodies against MyD88 (Abcam, ab219413), NF-κB (Abcam, ab288751), TNF-α (Abcam, ab183896), IL-1β (Abcam, ab234437), and IL-6 (Abcam, ab259341) at 4°C overnight and probed with secondary antibodies conjugated with horseradish peroxidase for 1-2 h at room temperature.

Techniques: Expressing, Western Blot, Staining, Flow Cytometry

Journal: iScience

Article Title: Hydrochlorothiazide-induced glucose metabolism disorder is mediated by the gut microbiota via LPS-TLR4-related macrophage polarization

doi: 10.1016/j.isci.2023.107130

Figure Lengend Snippet:

Article Snippet: After blocking with QuickBlock Western blocking buffer (Beyotime Biotechnology), the membrane was incubated with primary antibodies against MyD88 (Abcam, ab219413), NF-κB (Abcam, ab288751), TNF-α (Abcam, ab183896), IL-1β (Abcam, ab234437), and IL-6 (Abcam, ab259341) at 4°C overnight and probed with secondary antibodies conjugated with horseradish peroxidase for 1-2 h at room temperature.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay