Journal: Biomolecules

Article Title: Intestinal Barrier in Post- Campylobacter jejuni Irritable Bowel Syndrome

doi: 10.3390/biom13030449

Figure Lengend Snippet: EEA1 and cytokine intensity grade (0–4) at surface cells in the colon mucosa of PI-IBS patients and controls.

Article Snippet: Briefly, 3 µm paraffin sections were deparaffinized followed by retrieval of the antigenic epitopes via treatment in a pressure cooker in citrate buffer pH 6 for 3 min. Unspecific bindings were blocked by a serum-free protein block (DAKO, Glosdrup, Denmark) for 15 min at room temperature and the following polyclonal primary antibodies—rabbit-anti-human-EEA1 (abcam, Cambridge, UK), IL-1β (Bioss, Woburn, MA, USA), IL-22 (abcam), IL-4 (Tebubio, Frankfurt, Germany), IL-10 and IFNγ (both Peprotech, Hamburg, Germany), and goat-anti-human-IL-6 (R&D systems, Minneapolis, MN, USA) and -TNFα (PeproTech)—were applied in dilutions previously titrated in antibody diluent (DAKO) at 4 °C overnight.

Techniques:

Journal: Biomolecules

Article Title: Intestinal Barrier in Post- Campylobacter jejuni Irritable Bowel Syndrome

doi: 10.3390/biom13030449

Figure Lengend Snippet: Immunohistochemistry of Th1 cytokines (red) in the colon mucosa of controls and PI-IBS patients of IL-1β ( a , b ), IL-6 ( c , d ), IL-22 ( e , f ), INFγ ( g , h ) and TNFα ( i , j ), (counterstaining with hematoxylin = violet). Staining for IL-1β and IL-22 intensified in surface enterocytes in PI-IBS. Bar = 100 µm.

Article Snippet: Briefly, 3 µm paraffin sections were deparaffinized followed by retrieval of the antigenic epitopes via treatment in a pressure cooker in citrate buffer pH 6 for 3 min. Unspecific bindings were blocked by a serum-free protein block (DAKO, Glosdrup, Denmark) for 15 min at room temperature and the following polyclonal primary antibodies—rabbit-anti-human-EEA1 (abcam, Cambridge, UK), IL-1β (Bioss, Woburn, MA, USA), IL-22 (abcam), IL-4 (Tebubio, Frankfurt, Germany), IL-10 and IFNγ (both Peprotech, Hamburg, Germany), and goat-anti-human-IL-6 (R&D systems, Minneapolis, MN, USA) and -TNFα (PeproTech)—were applied in dilutions previously titrated in antibody diluent (DAKO) at 4 °C overnight.

Techniques: Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: An AhR-Ovol1-Id1 regulatory axis in keratinocytes promotes skin homeostasis against atopic dermatitis

doi: 10.1101/2024.01.29.577821

Figure Lengend Snippet: (A) RT-qPCR results of Ovol1 and Il1a expression in epidermal cells of the HDM/SEB-treated mice at day 7. n = 3 mice per group. (B) Experimental design for IL-1R Ab treatment in (C-K). Samples were harvested at day 11 after treatment for downstream analysis. IgG was used as a control. (C-D) Flow cytometry analysis of dermal γδT cells in the back skin of IgG- or IL-1R Ab-treated SSKO mice. See legends for additional details. n = 4 mice per group. (E) Representative photographs of IgG- or IL-1R Ab-treated SSKO mice. (F) Clinical scores of IgG- or IL-1R Ab-treated SSKO mice. n = 5 mice per group. (G) Representative skin histology (H/E staining). Scale bar = 100 μm. (H) Quantification of epidermal, dermal and total skin thickness in IgG- or IL-1R Ab-treated SSKO mice. n = 4 mice per group. (I) TEWL values of IgG- or IL-1R Ab-treated SSKO mice. n = 5 mice per group. (J-K) Flow cytometry analysis of the indicated cell types in the back skin of IgG- or IL-1R Ab-treated SSKO mice. See legends for additional details. n = 4 mice per group. (L) RT-qPCR results of Il4 , Il17a and Il33 expression in whole skin. Data are mean + SEM. n = 5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. p values were calculated using 2-tailed unpaired Student t test or Mann-Whitney U test.

Article Snippet: For IL-1R blocking, mice were i.p injected with 200 μg IL-1R antibody (BioXcell, Cat# BE0256, Clone JAMA-147) or IgG (BioXcell, Cat# BE0091) at days −1, 0, 3, 7 and 10 of HDM/SEB treatment.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: An AhR-Ovol1-Id1 regulatory axis in keratinocytes promotes skin homeostasis against atopic dermatitis

doi: 10.1101/2024.01.29.577821

Figure Lengend Snippet: Related to . (A-B) Percentages of different immune cell populations out of total skin cells (A) or skin CD45 + cells (B) in IgG- or IL-1R Ab-treated SSKO mice at day 11. n = 4 mice per group. (C) Clinical scores of skin eruption, scaling, bleeding and redness at day 11. n = 5 mice per group. (D) Quantification of dermal and total skin thickness. n = 4 mice per group. (E) Quantification of the indicated immune cell types per gram of skin tissue at day 11. n = 4 mice per group. (F) RT-qPCR analysis of the indicated genes in whole skin at day 11. n = 5 mice per group. (G) Quantification of the indicated immune cell types per gram of skin tissue in DMSO- or AGX51-treated SSKO mice at day 11. n = 4 mice per group. (H-I) Percentages of different immune cell populations out of total skin cells (H) or skin CD45 + cells (I) at day 11. n = 4-5 mice per group. (J) RT-qPCR results of the indicated genes in the whole skin at day 11. Data are mean + SEM. n = 4 mice per group. * p < 0.05, ** p < 0.01. p values were calculated using 2-tailed unpaired Student t test.

Article Snippet: For IL-1R blocking, mice were i.p injected with 200 μg IL-1R antibody (BioXcell, Cat# BE0256, Clone JAMA-147) or IgG (BioXcell, Cat# BE0091) at days −1, 0, 3, 7 and 10 of HDM/SEB treatment.

Techniques: Quantitative RT-PCR

Journal: PLOS ONE

Article Title: MICT ameliorates hypertensive nephropathy by inhibiting TLR4/NF-κB pathway and down-regulating NLRC4 inflammasome

doi: 10.1371/journal.pone.0306137

Figure Lengend Snippet: SHR-M group and SHR-H group performed exercise training with different exercise prescriptions for 8 weeks. (A) The weight of rats was measured every week. (B) Systolic blood pressure, (C) Diastolic blood pressure, (D) Mean arterial pressure and (E) Heart rate, were measured after 8-week exercise training. (F) Serum creatinine (Scr), (G) Blood urea nitrogen (BUN), and (H) Urine protein were measured to evaluate the renal function. *p< 0.05, **p< 0.01, ***p< 0.001.

Article Snippet: The primary antibodies used were anti-Collagen I (1:1000; Zenbio), anti-Collagen Ш (1:1000; Huabio), TGF-β1 (1:1000; Zenbio), α-SMA (1:1000; Zenbio), anti-TLR4 (1:1000; Proteintech), anti-NF-κB P65 (1:1000; Zenbio), anti-P-P65 (1:500; Zenbio), anti-IкBα (1:1000; Zenbio), and anti-p-IкBα (1:500; Zenbio), anti-NLRC4 (1:1000; AiFang), anti-Caspase-1 (1:1000; Zenbio), anti-ASC (1:1000; Zenbio), anti-IL-1β (1:500; Huabio), anti-GAPDH (1:50000; Proteintech).

Techniques:

Journal: PLOS ONE

Article Title: MICT ameliorates hypertensive nephropathy by inhibiting TLR4/NF-κB pathway and down-regulating NLRC4 inflammasome

doi: 10.1371/journal.pone.0306137

Figure Lengend Snippet: (A) Western blot was used to detect the protein expressions of (B) NLRC4, (C) Caspase-1, (D) ASC and (E) IL-1β in WKY group, SHR-S group, SHR-M group, SHR-H group. ELISA detection of (F) IL-6 and (G) TNF-α (H) IL-1β for evaluating the concentration of pro-inflammatory factors in serum. Data are presented as means ± SEM. N = 3 in each group. *p< 0.05, **p< 0.01.

Article Snippet: The primary antibodies used were anti-Collagen I (1:1000; Zenbio), anti-Collagen Ш (1:1000; Huabio), TGF-β1 (1:1000; Zenbio), α-SMA (1:1000; Zenbio), anti-TLR4 (1:1000; Proteintech), anti-NF-κB P65 (1:1000; Zenbio), anti-P-P65 (1:500; Zenbio), anti-IкBα (1:1000; Zenbio), and anti-p-IкBα (1:500; Zenbio), anti-NLRC4 (1:1000; AiFang), anti-Caspase-1 (1:1000; Zenbio), anti-ASC (1:1000; Zenbio), anti-IL-1β (1:500; Huabio), anti-GAPDH (1:50000; Proteintech).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLOS ONE

Article Title: MICT ameliorates hypertensive nephropathy by inhibiting TLR4/NF-κB pathway and down-regulating NLRC4 inflammasome

doi: 10.1371/journal.pone.0306137

Figure Lengend Snippet: (B) CCK-8 assay was used to determine the cell viability. We found that 10 μM Ang II significantly reduced the cell viability of HK-2 cells. We selected 10 μM Ang II for inducing HN model in vitro based on the results of CCK-8. HK-2 cells were treated with 1, 3, 5 μM TAK-242 for 2 hours prior to 10 μM Ang II for 24 hours. (A) Western blot was used to detect the protein levels of (C) TLR4, (D) p-p65 and p65, (E) p-IκBα and IκBα. Data are presented as means ± SEM. N = 3 in each group. *p< 0.05, **p< 0.01, ***p< 0.001.

Article Snippet: The primary antibodies used were anti-Collagen I (1:1000; Zenbio), anti-Collagen Ш (1:1000; Huabio), TGF-β1 (1:1000; Zenbio), α-SMA (1:1000; Zenbio), anti-TLR4 (1:1000; Proteintech), anti-NF-κB P65 (1:1000; Zenbio), anti-P-P65 (1:500; Zenbio), anti-IкBα (1:1000; Zenbio), and anti-p-IкBα (1:500; Zenbio), anti-NLRC4 (1:1000; AiFang), anti-Caspase-1 (1:1000; Zenbio), anti-ASC (1:1000; Zenbio), anti-IL-1β (1:500; Huabio), anti-GAPDH (1:50000; Proteintech).

Techniques: CCK-8 Assay, In Vitro, Western Blot

Journal: PLOS ONE

Article Title: MICT ameliorates hypertensive nephropathy by inhibiting TLR4/NF-κB pathway and down-regulating NLRC4 inflammasome

doi: 10.1371/journal.pone.0306137

Figure Lengend Snippet: HK-2 cells were treated with 1, 3, 5 μM TAK-242 for 2 hours prior to 10 μM Ang II for 24 hours. (A, B) Western blot was used to detect the protein levels of (C-F) fibrosis-related molecules and (G-J) NLRC4 inflammasome. Data are presented as means ± SEM. N = 3 in each group. *p< 0.05, **p< 0.01, ***p< 0.001.

Article Snippet: The primary antibodies used were anti-Collagen I (1:1000; Zenbio), anti-Collagen Ш (1:1000; Huabio), TGF-β1 (1:1000; Zenbio), α-SMA (1:1000; Zenbio), anti-TLR4 (1:1000; Proteintech), anti-NF-κB P65 (1:1000; Zenbio), anti-P-P65 (1:500; Zenbio), anti-IкBα (1:1000; Zenbio), and anti-p-IкBα (1:500; Zenbio), anti-NLRC4 (1:1000; AiFang), anti-Caspase-1 (1:1000; Zenbio), anti-ASC (1:1000; Zenbio), anti-IL-1β (1:500; Huabio), anti-GAPDH (1:50000; Proteintech).

Techniques: Western Blot

Journal: iScience

Article Title: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis

doi: 10.1016/j.isci.2022.105121

Figure Lengend Snippet: Inflammasome signaling downstream of MLKL confers host resistance to C. perfringens infection WT and Mlkl −/− mice were intramuscularly infected with C. perfringens (1 × 10 7 CFU, n = 10 each group) for 24 h (A) The muscle tissue lysate was analyzed for Caspase-1, IL-1β, and ASC by Western blotting. GAPDH was used as a loading control. (D and G) The homogenate supernatant of the muscle tissue was analyzed for the presence of IL-18, IL-1β, and TNF-α protein using ELISA. (M) Bacterial load in the muscle was detected. D-4-Amino-3-isoxazolidinone-pretreated WT and Mlkl −/− mice were orally infected with C. perfringens (1 × 10 8 CFU, n = 10 each group) for 24 h (B and C) Duodenal and cecal tissues were collected and homogenized, and then immunoblotting for Caspase-1, IL-1β, ASC, and GAPDH, respectively. (E, F, H, and I) Levels of IL-18, IL-1β, and TNF-α in duodenum or cecum were determined by ELISA. For one group of Mlkl −/− mice, 1.0 μg recombinant IL-18 was injected intraperitoneally daily starting the day prior to the intramuscularly or orally challenged with C. perfringens (n = 10 each group, at 24 h p.i.). (J) Representative H&E staining and pathologic photographs of the muscle tissue (1 × 10 7 CFU, left panel, magnification ×100, right panel, magnification, ×400). (K and L) Representative H&E staining and pathologic photographs of the duodenum and cecum tissues (1 × 10 8 CFU, left panel, magnification ×100, right panel, magnification, ×200). Bacterial burdens in the duodenum (N), cecum (O), MLN (P), liver (Q), and spleen (R) were examined. Data were considered significant when ∗∗p < 0.01.

Article Snippet: The membranes were blotted with primary antibodies against Caspase-1 (Adipogen, #A28881708), IL-1β (Adipogen, #PRP1119-20μg), ASC (Adipogen, #A29151803f), GSDMD (Santa Cruz, #sc-393656), P38 MAPK (ABclonal, #A4771), p-P38 MAPK (ABclonal, #AP0526), JNK (Proteintech, #66210-1-lg), p-JNK (ABclonal, #AP0276), ERK1/2 (ABclonal, #A10613), p-ERK1/2 (ABclonal, #AP0472), and GADPH (Roteintech, #60004-1-Ig).

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Injection, Staining

Journal: iScience

Article Title: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis

doi: 10.1016/j.isci.2022.105121

Figure Lengend Snippet: Involvement of the NLRP3 inflammasome in MLKL-mediated host defense WT and Mlkl −/− mice were intramuscularly infected with C. perfringens (1 × 10 7 CFU) for 24 h (A) Representative muscle sections. p -MLKL (IHC) stained brown (upper panel, magnification ×100, lower panel, magnification, ×400). D-4-Amino-3-isoxazolidinone-pretreated WT and Mlkl −/− mice were orally infected with C. perfringens (1 × 10 8 CFU) for 24 h (B and C) Representative duodenum and cecum sections. p -MLKL (IHC) stained brown (upper panel, magnification ×100, lower panel, magnification, ×200). LPS-primed WT and Nlrp3 −/− BMDMs were stimulated with ATP (5mM, 30min), C. perfringens strain ATCC13124, CP1 or CP3 (MOI = 20, 90 min). (D) Cell supernatants and cell extracts immunoblotted for Caspase-1, IL-1β and ASC. GAPDH served as loading controls. (E and F) Culture supernatants were analyzed for IL-1β, IL-18 and TNF-α by ELISA. (G) LDH release was quantified to monitor cell lysis. Data are shown as the percentage of LDH release by Triton X-100 treated control cells. (H) Cell supernatants and cell extracts immunoblotted for Caspase-1, GSDMD, IL-1β, ASC, and GAPDH. (I and J) Culture supernatants were analyzed for IL-1β, IL-18, and TNF-α by ELISA. (K) LDH release was quantified to monitor cell lysis. Data are shown as the percentage of LDH release by Triton X-100 treated control cells. LPS-primed WT and Mlkl −/− BMDMs were stimulated with ATP (5mM, 30min), C. perfringens strain ATCC13124, CP1, or CP3 (MOI = 20, 90 min). (L) Cell supernatants and cell extracts immunoblotted for Caspase-1, GSDMD, IL-1β, ASC, p -MLKL, and GAPDH. (M and N) Culture supernatants were analyzed for IL-1β, IL-18 and TNF-α by ELISA. (O) LDH release was quantified to monitor cell lysis. Data are shown as the percentage of LDH release by Triton X-100 treated control cells. LPS-primed WT, Mlkl −/− and Nlrp3 −/− BMDMs were stimulated with ATP (5mM, 30min), C. perfringens strain ATCC13124, CP1, or CP3 (MOI = 20, 90 min). (P and Q) The cells were fixed, permeabilized, and stained for ASC (green). DAPI was used to label nuclei (blue). magnification, ×400. The percentage of cells containing ASC speckles was quantified. LPS-primed WT and Mlkl −/− BMDMs were stimulated with live bacteria (MOI = 20, 90 min), heat-killed bacteria (HK, MOI = 20, 90 min), bacterial culture supernatants (SN, 5 h) or bacterial α-toxin (20 μg/mL, 5 h). (R and T) Culture supernatants were analyzed for IL-1β, IL-18, and TNF-α by ELISA. (U–W) To determine the bacterial killing capacity of BMDMs, LPS-primed WT, Mlkl −/− , Nlrp3 −/− , or Caspase-1/11 −/− BMDMs were incubated with rIL-18 (1000 pg/mL) or PBS for 1 h before were infected with C. perfringens strain ATCC13124, CP1 or CP3 (MOI = 5) for 6 h, the supernatants were collected and plated on BHI agar plates to enumerate the bacteria after 24 h of anaerobic culture. Graphs are means ± SD from data pooled from four to six (E, F, G, I, J, K, M, N, O, Q, R, S, T, U, V, and W) biological replicates. Data were considered significant when ∗p < 0.05 or ∗∗p < 0.01.

Article Snippet: The membranes were blotted with primary antibodies against Caspase-1 (Adipogen, #A28881708), IL-1β (Adipogen, #PRP1119-20μg), ASC (Adipogen, #A29151803f), GSDMD (Santa Cruz, #sc-393656), P38 MAPK (ABclonal, #A4771), p-P38 MAPK (ABclonal, #AP0526), JNK (Proteintech, #66210-1-lg), p-JNK (ABclonal, #AP0276), ERK1/2 (ABclonal, #A10613), p-ERK1/2 (ABclonal, #AP0472), and GADPH (Roteintech, #60004-1-Ig).

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay, Lysis, Incubation

Journal: iScience

Article Title: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis

doi: 10.1016/j.isci.2022.105121

Figure Lengend Snippet:

Article Snippet: The membranes were blotted with primary antibodies against Caspase-1 (Adipogen, #A28881708), IL-1β (Adipogen, #PRP1119-20μg), ASC (Adipogen, #A29151803f), GSDMD (Santa Cruz, #sc-393656), P38 MAPK (ABclonal, #A4771), p-P38 MAPK (ABclonal, #AP0526), JNK (Proteintech, #66210-1-lg), p-JNK (ABclonal, #AP0276), ERK1/2 (ABclonal, #A10613), p-ERK1/2 (ABclonal, #AP0472), and GADPH (Roteintech, #60004-1-Ig).

Techniques: Recombinant, Protease Inhibitor, SYBR Green Assay, Isolation, Staining, LDH Cytotoxicity Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Software