Journal: Scientific Reports

Article Title: Pollen/TLR4 Innate Immunity Signaling Initiates IL-33/ST2/Th2 Pathways in Allergic Inflammation

doi: 10.1038/srep36150

Figure Lengend Snippet: ( A ) The stimulated mRNA expression of IL-33/ST2 signaling molecules and Th2 cytokines by corneal epithelium and conjunctiva in SRW-EAC BALB/c mice, evaluated by RT-qPCR. ( B ) Increased IL-33 protein levels in conjunctiva of EAC mice by ELISA. ( C ) Increased IL-33 production in ocular epithelium of EAC mice by immunohistochemical staining. Results shown are the Mean ± SD of four independent experiments. * P < 0.05, **P < 0.01, ***P < 0.001 , n = 4. ND: not detected.

Article Snippet: Mouse ELISA kits for IL-4, IL-5 and IL-13 were from BosterBio (Pleasanton, CA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Pollen/TLR4 Innate Immunity Signaling Initiates IL-33/ST2/Th2 Pathways in Allergic Inflammation

doi: 10.1038/srep36150

Figure Lengend Snippet: ( A,C ) Immunohistochemical staining of IL-33 and its signaling molecules and Th2 cytokines on cornea and conjunctiva (Conj) in wild type BALB/c and Tlr4-d mice ( A ), as well as in MyD88 +/+ and MyD88 − / − mice ( C ), challenged by SRW pollen, with PBS-treated mice as controls. Bar: 20 μm; Arrows: positive staining signals. ( B,D ) Protein levels of Th2 cytokines by ELISA in conjunctiva of wild type BALB/c and Tlr4-d mice ( B ), as well as in MyD88 +/+ and MyD88 − / − mice ( D ). Results shown are Mean ± SD. *P < 0.05, **P < 0.01 ; n = 4, compared with PBS controls.

Article Snippet: Mouse ELISA kits for IL-4, IL-5 and IL-13 were from BosterBio (Pleasanton, CA).

Techniques: Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Pollen/TLR4 Innate Immunity Signaling Initiates IL-33/ST2/Th2 Pathways in Allergic Inflammation

doi: 10.1038/srep36150

Figure Lengend Snippet: ( A ) The upregulated mRNA of Th2 cell surface markers and cytokines by CLNs, evaluated by RT-qPCR. ( B,C ) Protein levels of ST2 and Th2 cytokines in CLNs by ELISA. ( D ) Immunofluorescent staining of Th2 cell markers and cytokines in CLNs. ( E,F ) Direct effects of IL-33 on Th2 cytokine expression by isolated CD4 + T cells at mRNA ( E ) and protein levels ( F ). Results shown are Mean ± SD. *P < 0.05, **P < 0.01 ***P < 0.001 ; n = 4, compared with PBS or untreated (UT) controls.

Article Snippet: Mouse ELISA kits for IL-4, IL-5 and IL-13 were from BosterBio (Pleasanton, CA).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Isolation

Journal: Scientific Reports

Article Title: Pollen/TLR4 Innate Immunity Signaling Initiates IL-33/ST2/Th2 Pathways in Allergic Inflammation

doi: 10.1038/srep36150

Figure Lengend Snippet: ( A,B ) BALB/c mice were topically instilled with SRWe at 150 μg/5 μl/eye without or with pre-instilled rat anti-mouse TLR4 antibody (1 μg/5 μl/eye) or its isotype rat IgG2a, and LPS at 5 μg/5 μl/eye or 0.1 μg/5 μl/eye was used as positive control. ( C,D ) IL-33 induction by topically challenged SRWe in wild type BALB/C and Tlr4-d mice. ( E,F ) IL-33 induction by topically challenge in MyD88 +/+ and MyD88 − / − mice. ( A,C,E ) Show IL-33 mRNA expression by RT-qPCR, and ( B,D,F ) show its protein levels by ELISA. Results shown are Mean ± SD (n = 5). *P < 0.05, **P < 0.01, compared with PBS controls.

Article Snippet: Mouse ELISA kits for IL-4, IL-5 and IL-13 were from BosterBio (Pleasanton, CA).

Techniques: Positive Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: Staphylococcus aureus increases Prostaglandin E 2 secretion in cow neutrophils by activating TLR2, TLR4, and NLRP3 inflammasome signaling pathways

doi: 10.3389/fmicb.2023.1163261

Figure Lengend Snippet: Staphylococcus aureus lipoprotein is an important protein that can induce the secretion of PGE 2 and cytokines by neutrophils in dairy cows and can also activate TLR2, TLR4, and NLRP3 receptors and their mediated MAPK and Caspase-1 signaling pathways. ELISA results showed the secretion levels of cytokines IL-1β (A) , IL-6 (B) , IL-10 (C) , IL-8 (D) , and PGE 2 (E) in the uninfected, SA113, and Δ lgt groups after neutrophil infection with MOI 3:1 and MOI 10:1. Western blot analysis revealed the activation of the MAPK (F–H) and Caspase-1 (I, J) signaling pathways. TLR2, TLR4, and NLRP3 receptor genes (K–M) and protein (N–Q) expressions were detected by qRT-PCR and Western blot. ns P > 0.05, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Fetal bovine serum (FBS), RPMI1640 medium, and PBS (Hyclon, Logen, UT); dairy cow peripheral blood neutrophil isolation kit (Hao Yang, Tian Jing, China); MH broth (Oxoid, Hampshire, UK); TLR2 receptor inhibitor (C29, MCE, China), TLR4 receptor inhibitor (TAK-242, MCE, China), NLRP3 receptor inhibitor (MCC950, MCE, China); COX-2 inhibitor (CAY10404), mPGES-1 inhibitor (MF63) (Cayman Chemical, MI, United States); gentamicin sulfate(Promega, Wisconsin, United States); Bovine IL-1β, IL-10, IL-8 ELISA Kits (Kingfisher, Biotech); Bovine IL-6 ELISA Kit (R&D Systems, California, United States); PGE 2 ELISA Kit (Cayman Chemical, MI, United States); M-PER mammalian protein extraction reagent, Halt protease inhibitor, pre-stained protein ladders, blocking buffer, starting block T20 (TBS), LIVE/DEAD Bac Light Bacterial Viability Kits (Thermo Fisher Scientific); SDS-PAGE loading buffer (Takara, Shiga, Japan); Pierce BCA Protein Assay Kit (Rockford, United States); SDS-PAGE gel electrophoresis kit (Solarbio, Beijing, China); Axy Prep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, United States); Primer Script RT Master Mix (Takara); SYBR Green Master (Rox) (Roche, Basel, Switzerland).

Techniques: Protein-Protein interactions, Enzyme-linked Immunosorbent Assay, Infection, Western Blot, Activation Assay, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Staphylococcus aureus increases Prostaglandin E 2 secretion in cow neutrophils by activating TLR2, TLR4, and NLRP3 inflammasome signaling pathways

doi: 10.3389/fmicb.2023.1163261

Figure Lengend Snippet: The secretion of PGE 2 and cytokines mediated by neutrophils depends on TLR2, TLR4, and NLRP3 receptors. ELISA results showed the secretion levels of cytokines IL-1β (A) , IL-6 (B) , IL-8 (C) , IL-10 (D) , and PGE2 (E) in the SA113, C29, TAK-242, and MCC950 groups after neutrophils infection with MOI 3:1 and MOI 10:1. Western blot analysis revealed the activation of the MAPK (F, G, I) and Caspase-1 (H, J) signaling pathways. ns P > 0.05, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Fetal bovine serum (FBS), RPMI1640 medium, and PBS (Hyclon, Logen, UT); dairy cow peripheral blood neutrophil isolation kit (Hao Yang, Tian Jing, China); MH broth (Oxoid, Hampshire, UK); TLR2 receptor inhibitor (C29, MCE, China), TLR4 receptor inhibitor (TAK-242, MCE, China), NLRP3 receptor inhibitor (MCC950, MCE, China); COX-2 inhibitor (CAY10404), mPGES-1 inhibitor (MF63) (Cayman Chemical, MI, United States); gentamicin sulfate(Promega, Wisconsin, United States); Bovine IL-1β, IL-10, IL-8 ELISA Kits (Kingfisher, Biotech); Bovine IL-6 ELISA Kit (R&D Systems, California, United States); PGE 2 ELISA Kit (Cayman Chemical, MI, United States); M-PER mammalian protein extraction reagent, Halt protease inhibitor, pre-stained protein ladders, blocking buffer, starting block T20 (TBS), LIVE/DEAD Bac Light Bacterial Viability Kits (Thermo Fisher Scientific); SDS-PAGE loading buffer (Takara, Shiga, Japan); Pierce BCA Protein Assay Kit (Rockford, United States); SDS-PAGE gel electrophoresis kit (Solarbio, Beijing, China); Axy Prep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, United States); Primer Script RT Master Mix (Takara); SYBR Green Master (Rox) (Roche, Basel, Switzerland).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Western Blot, Activation Assay, Protein-Protein interactions

Journal: Frontiers in Microbiology

Article Title: Staphylococcus aureus increases Prostaglandin E 2 secretion in cow neutrophils by activating TLR2, TLR4, and NLRP3 inflammasome signaling pathways

doi: 10.3389/fmicb.2023.1163261

Figure Lengend Snippet: PGE 2 can affect the secretion of pro-inflammatory factors, anti-inflammatory factors, and chemokines in neutrophils of cows infected with S. aureus . ELISA results showed that IL-1β (A) , IL-6 (B) , IL-8 (C) , IL-10 (D) , and PGE 2 (E) were secreted in the SA113 group, the uninfected group, the CAY10404 group, and the MF63 group. ns P > 0.05, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Fetal bovine serum (FBS), RPMI1640 medium, and PBS (Hyclon, Logen, UT); dairy cow peripheral blood neutrophil isolation kit (Hao Yang, Tian Jing, China); MH broth (Oxoid, Hampshire, UK); TLR2 receptor inhibitor (C29, MCE, China), TLR4 receptor inhibitor (TAK-242, MCE, China), NLRP3 receptor inhibitor (MCC950, MCE, China); COX-2 inhibitor (CAY10404), mPGES-1 inhibitor (MF63) (Cayman Chemical, MI, United States); gentamicin sulfate(Promega, Wisconsin, United States); Bovine IL-1β, IL-10, IL-8 ELISA Kits (Kingfisher, Biotech); Bovine IL-6 ELISA Kit (R&D Systems, California, United States); PGE 2 ELISA Kit (Cayman Chemical, MI, United States); M-PER mammalian protein extraction reagent, Halt protease inhibitor, pre-stained protein ladders, blocking buffer, starting block T20 (TBS), LIVE/DEAD Bac Light Bacterial Viability Kits (Thermo Fisher Scientific); SDS-PAGE loading buffer (Takara, Shiga, Japan); Pierce BCA Protein Assay Kit (Rockford, United States); SDS-PAGE gel electrophoresis kit (Solarbio, Beijing, China); Axy Prep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, United States); Primer Script RT Master Mix (Takara); SYBR Green Master (Rox) (Roche, Basel, Switzerland).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Identification of a P62-TIF-IA axis that drives nucleolar fusion and the senescence associated secretory phenotype

doi: 10.1101/2023.12.05.570133

Figure Lengend Snippet: (A) The levels of TIF-1A transcription were investigated by qRT-PCR in IMR90 ER:Stop and ER:Ras cells exposed to 4-OHT for the times indicated in hours (n=3). (B) HCT116 cells were treated with DMSO (control) or etoposide for 8h. Whole cell lysates were immunoprecipitated with IgG or TIF-IA antibody then precipitated proteins analysed by quantitative mass spectrometry. Volcano plot shows a comparison of IgG v TIF-IA for DMSO treated cells. Blue dot=non-significant. Red dot=significant change (fold change (FC) >2, P<0.05). (C) Immmunomicrograph showing p62-TIF-IA co-localisation in cytoplasmic foci in IMR90 cells. (D) Top: Immunomicrograph of IMR90 cells treated with the lysosome inhibitor, bafilomycin A (BafA1) or DMSO carrier. Bottom: Cell profiler was used to quantify cytoplasmic and nuclear TIF-IA intensity. A minimum of 50 cells were analysed/treatment for each biological repeat. N=2. (E) Representative immunomicrographs (63X) showing level and localisation of TIF-IA and p62 in wild type (WT) A549 cells and those in which the autophagy gene, atg5, has been deleted (ΔATG5). White arrow indicates co-localised foci in WT cells. N=5. (F) Immunoblot performed on whole cell extracts from WT and Δatg5 cells. n=5 (G) Immunoblot showing levels of TIF-IA and p62 in whole cell lysates following siRNA depletion of p62. n=2. (H) TIF-IA was immunoprecipitated (IP) from WT or Δatg5 A549 cells then recovered proteins analysed by immunoblot (IB) for p62. Stripped gels were re-probed for TIF-IA. Input levels of protein are shown. Rabbit IgG acts as a control. n=3 (I) P62 binds to a high molecular weight (HMW) form of TIF-IA in a manner dependent on the dimerization and ubiquitin binding domains. Wild type and Δatg5 A549 cells were transfected with plasmids expressing GFP-P62 WT, GFP-p62 ΔPB1 (deleted for the PB1 domain required for dimerization) or GFP-p62ΔUBA (deleted for the ubiquitin binding domain). GFP-tagged proteins were immuoprecipitated (IP) from whole-cell lysates using GFP-TRAP beads. Precipitated proteins were subjected to immunoblotting (IB) for TIF-IA and GFP. Input levels of TIF-IA are shown. N=2. Actin is used as a loading control throughout.

Article Snippet: Primary antibodies used were: TIF-IA (rabbit, 1:1000, BioAssayTech B8433); Rrn3 (Mouse, 1:500, Santa Cruz sc-390464); p62 (Mouse, 1:1000 BD Transduction Labs BD610833); GFP (Rabbit, 1:1000 Santa Cruz, sc-8433); Actin-HRP (Mouse 1:2000 sc-47778 Santa Cruz).

Techniques: Quantitative RT-PCR, Control, Immunoprecipitation, Mass Spectrometry, Comparison, Western Blot, High Molecular Weight, Ubiquitin Proteomics, Binding Assay, Transfection, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Small intestinal eosinophils regulate Th17 cells by producing IL-1 receptor antagonist

doi: 10.1084/jem.20141388

Figure Lengend Snippet: Small intestinal eosinophils express high levels of the IL-1Ra. (A) Microarray analysis of DCs and eosinophils (EO) from the small intestine of WT C57BL/6 mice. (B) Western blot analysis of IL-1Ra protein in cell lysates of lymphocytes (Lym), DCs, and eosinophils sorted from the small intestinal LP (left). (Right) IL-1Ra protein detected from whole-cell lysates of spleen (SP), MLNs, PPs, and small intestinal LP (SI-LP). (C) Western blot analysis of IL-1Ra protein in lysates of total small intestinal tissue (jejunum) and small intestinal epithelial cells (SI-IEC) purified from BALB/c (WT) or ΔdblGATA-1 (KO) mice. (D) IL-1Ra secreted by eosinophils (10 6 ) or other cells (Non-EO; 10 6 ) in small intestine during a 24-h culture was measured by ELISA. (E) Various cytokines secreted by sorted small intestinal eosinophils (10 6 ) during a 24-h culture were measured by ELISA. (F) Quantitative RT-PCR analysis of IL-1Ra in eosinophils isolated from bone marrow, blood, or small intestine. (G) IL-1Ra in culture supernatants of eosinophils (4 × 10 4 ) sorted from bone marrow, blood, or small intestine was measured by ELISA. (H) IL-1Ra secreted by eosinophils (10 5 ) sorted from small intestine, normal lung, or allergic asthma-induced lung during a 24-h culture was measured by ELISA. Data are representative of two (E and H) or at least three (B–D, F, and G) experiments. Bar graphs show the mean ± SEM. ND, not detected. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test).

Article Snippet: Mouse ELISA kits (IL-1Ra/IL-1F3; R&D Systems; IL-1β, IL-4, IL-5, IL-6, IL-13, IL-12p40, and IL-17A; eBioscience; TNF; BD) were used according to the manufacturer’s instructions to measure these cytokine levels in mouse serum or cell culture supernatants.

Techniques: Microarray, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Small intestinal eosinophils regulate Th17 cells by producing IL-1 receptor antagonist

doi: 10.1084/jem.20141388

Figure Lengend Snippet: Eosinophils secrete IL-1Ra upon GM-CSF stimulation. (A and B) IL-1Ra in the culture supernatant of small intestinal eosinophils sorted from MyD88-deficient (KO; 10 5 ) or littermate control (WT; 10 5 ) mice (A) and SPF (5 × 10 4 ) or GF (5 × 10 4 ) mice (B) after a 24-h culture was measured by ELISA. (C) IL-1Ra in the culture supernatant of bone marrow eosinophils (2 × 10 4 ) after a 3 or 5 d culture with acetate (1 mM), butyrate (1 mM), or propionate (1 mM) was measured by ELISA. (D) IL-1Ra in culture supernatant of eosinophils (5 × 10 4 ) sorted from small intestine or large intestine was measured by ELISA. (E) IL-1Ra in the culture supernatant of bone marrow eosinophils (2 × 10 4 ) after a 3 or 5 d culture with GM-CSF (10 ng/ml), IL-5 (10 ng/ml), IL-33 (10 ng/ml), TGF-β (5 ng/ml), or retinoic acid (RA; 10 mM) was measured by ELISA. (F) IL-1Ra in the culture supernatant of bone marrow eosinophils (1.5 × 10 4 ) after a 5 d culture with GM-CSF (10 ng/ml) in the presence of GM-CSF receptor α blocking antibodies (α-GM-CSFRα; 8 µg/ml) or isotype control (Rat IgG2a; 8 µg/ml) was measured by ELISA. (G) Quantitative RT-PCR analysis of GM-CSF in bone marrow, blood, or jejunum (SI). (H) Quantitative RT-PCR analysis of GM-CSF receptor in eosinophils sorted from bone marrow, blood, or small intestine. All data are representative of at least three independent experiments. Bar graphs show the mean ± SEM. *, P < 0.05, ***, P < 0.001; NS, not significant (unpaired Student’s t test).

Article Snippet: Mouse ELISA kits (IL-1Ra/IL-1F3; R&D Systems; IL-1β, IL-4, IL-5, IL-6, IL-13, IL-12p40, and IL-17A; eBioscience; TNF; BD) were used according to the manufacturer’s instructions to measure these cytokine levels in mouse serum or cell culture supernatants.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Blocking Assay, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Small intestinal eosinophils regulate Th17 cells by producing IL-1 receptor antagonist

doi: 10.1084/jem.20141388

Figure Lengend Snippet: An antiinflammatory function of eosinophils. (A) IL-1Ra in serum from ΔdblGATA-1 or littermate control mice (WT) was measured by ELISA. Results shown are representative of at least three independent experiments ( n ≥ 4 mice per group per experiment). Bar graphs show the mean ± SEM. *, P < 0.05 (unpaired Student’s t test). (B) Mice were intraperitoneally injected with 15 mg/kg of LPS. IL-1Ra–hybrid Fc fusion protein (IL-1Ra–Fc; 25 mg/kg) was injected 4 h before and 20 min after the injection of LPS. Thereafter, mice were treated four times (every 3 h) with IL-1Ra–Fc. Survival of mice was monitored for 7 d (WT, n = 10; WT + IL-1Ra–Fc, n = 9; ΔdblGATA-1, n = 10; ΔdblGATA-1 + IL-1Ra–Fc, n = 10). Data are representative of three similar experiments. Statistical curve comparison was analyzed by Log-rank (Mantel-Cox) test. *, P < 0.05; ***, P < 0.001. (C) Serum samples of mice in B were collected at 6 h after LPS injection and cytokines were measured. Data are representative of two independent experiments. Bar graphs show the mean ± SEM. ND, not detected. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test).

Article Snippet: Mouse ELISA kits (IL-1Ra/IL-1F3; R&D Systems; IL-1β, IL-4, IL-5, IL-6, IL-13, IL-12p40, and IL-17A; eBioscience; TNF; BD) were used according to the manufacturer’s instructions to measure these cytokine levels in mouse serum or cell culture supernatants.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Injection, Comparison

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Expression of insulin‐like growth factor‐binding protein 5 (IGFBP5) is elevated in ischaemic tissue or hypoxic human umbilical vein endothelial cells (HUVECs). (A) Representative immunofluorescence images and (B) quantification of the expression of IGFBP5 in ischaemic or non‐ischaemic tissues from patients with critical limb ischaemia ( n = 6). (C) Representative immunofluorescence images and (D) quantification of the expression of IGFBP5 in gastrocnemius of the ischaemic hindlimb of mice ( n = 5). (E) mRNA expression of IGFBP1‐7 tested by qRT‐PCR in HUVECs under hypoxia stimulation for 24 h ( n = 6). (F) Representative Western blotting images and quantification of the expression of IGFBP5 in HUVECs under hypoxia stimulation for 24 h. (G) Representative immunofluorescence images and (H) quantification of the expression of IGFBP5 in HUVECs under hypoxia stimulation for 24 h ( n = 6).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Expressing, Binding Assay, Immunofluorescence, Quantitative RT-PCR, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Endothelial cell (EC)‐specific deletion of insulin‐like growth factor‐binding protein 5 (IGFBP5) attenuates the damage of the ischaemic hindlimb in mice. (A) Representative blood flow images of laser Doppler‐based tissue perfusion system in CDH5‐Cre‐IGFBP5 flox/flox (IGFBP5 EKO ) and IGFBP5 flox/flox (IGFBP5 f/f ) mice after ischaemia induction (0 W) and 4 weeks after ischaemia. (B) Quantification of blood flow in the hindlimb of IGFBP5 EKO and IGFBP5 f/f mice before (−1 D) and after ischaemia induction at 0, 1, 2, 3 and 4 weeks ( n = 8 in each group). ### p < .001 compared to IGFBP5 f/f ‐sham; * p < .05, ** p < .01 and *** p < .001 compared to IGFBP5 f/f ‐HLI. (C) Percentage of limb salvage, foot necrosis and limb loss in IGFBP5 EKO and IGFBP5 f/f mice treated with sham or hindlimb ischaemia (HLI) ( n = 8 in each group). (D) Representative immunofluorescence staining images and (E) quantification of CD31 (red) in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (F) Representative haematoxylin‒eosin and picrosirius red staining images and (G) quantification of fibrosis area in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (H) Representative immunofluorescence staining images and (I) quantification of dihydroethidium (DHE) staining for reactive oxygen species (ROS) in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (J) Representative immunofluorescence staining images and (K) quantification of TUNEL staining in gastrocnemius of hindlimb from IGFBP5 EKO and IGFBP5 f/f mice treated with sham or HLI ( n = 5 in each group). (L) Representative microscopy images of aortic sprouting assay and (M) quantification of tube length of the aorta from IGFBP5 EKO and IGFBP5 f/f mice at 4 and 7 days after being placed in plates with Matrigel. (N) Representative images and (O) quantification of cutaneous wound healing in IGFBP5 EKO and IGFBP5 f/f mice at 0, 4 and 7 days after wound induction ( * p < .05, *** p < .001 vs. IGFBP5 f/f ).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Immunofluorescence, Staining, TUNEL Assay, Microscopy

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Insulin‐like growth factor‐binding protein 5 (IGFBP5) knockdown promotes tube formation, cell proliferation and migration in human umbilical vein endothelial cells (HUVECs). (A) Representative heatmap, (B) volcano plot and (C) Gene Ontology (GO) biological process enrichment pathway in IGFBP5 shRNA (sh‐IGFBP5)‐ or control shRNA (sh‐NC)‐transfected HUVECs. (D) Representative images and (E) quantification of tube formation in sh‐NC‐ and sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (F) Representative immunofluorescence images and (G) quantification of 5‐ethynyl‐2′‐deoxyuridine (EdU, green)‐stained HUVECs transfected with sh‐NC or sh‐IGFBP5 ( n = 5 in each group). (H) Representative images and (I) quantification of flow cytometry for cell cycle in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (J) Representative images and (K) quantification of wound healing assay in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (L) Representative images and (M) quantification of transwell assay in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 5 in each group). (N) Representative Western blotting images and (O) quantification of IGFBP5, p‐IGF1R and IGF1R expression in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs. (P) Representative Western blotting images and (Q) quantification of p‐Erk1/2, Erk1/2, p‐Akt and Akt expression in sh‐NC‐ or sh‐IGFBP5‐transfected HUVECs ( n = 6 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Knockdown, Migration, shRNA, Control, Transfection, Immunofluorescence, Staining, Flow Cytometry, Wound Healing Assay, Transwell Assay, Western Blot, Expressing

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Recombinant human insulin‐like growth factor‐binding protein 5 (IGFBP5) suppresses tube formation, cell proliferation and migration. (A) Representative images and (B) quantification of tube formation in recombinant human IGFBP5 (rhIGFBP5)‐ or vehicle‐treated human umbilical vein endothelial cells (HUVECs). (C) Representative immunofluorescence images and (G) quantification of 5‐ethynyl‐2′‐deoxyuridine (EdU, green)‐stained HUVECs treated with rhIGFBP5 or vehicle ( n = 5 in each group). (E) Representative images and (F) quantification of flow cytometry for cell cycle in rhIGFBP5‐ or vehicle‐treated HUVECs ( n = 5 in each group). (G) Representative images and (H) quantification of wound healing assay in rhIGFBP5‐ or vehicle‐treated HUVECs. (I) Representative images and (J) quantification of transwell assay in rhIGFBP5‐ or vehicle‐treated HUVECs ( n = 5 in each group). Representative Western blotting images and quantification of (K) p‐IGF1R and IGF1R, (L) p‐Erk1/2 and Erk1/2 and (M) p‐Akt, and Akt in rhIGFBP5‐ or vehicle‐treated HUVECs ( n = 6 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Recombinant, Binding Assay, Migration, Immunofluorescence, Staining, Flow Cytometry, Wound Healing Assay, Transwell Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Recombinant human insulin‐like growth factor‐binding protein 5 (IGFBP5) restrains IGF1‐ or IGF2‐induced tube formation, cell proliferation and migration. (A) Representative tube formation images and quantification of human umbilical vein endothelial cells (HUVECs) treated with rhIGF1 in the presence or absence of recombinant human IGFBP5 (rhIGFBP5) ( n = 5 in each group). (B) Representative tube formation images and quantification of HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (C) Representative immunofluorescence images and quantification of 5‐ethynyl‐2′‐deoxyuridine (EdU, green)‐stained HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (D) Representative immunofluorescence images and quantification of EdU (green)‐stained HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (E) Representative images and quantification of flow cytometry for cell cycle of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (F) Representative images and quantification of flow cytometry for cell cycle of HUVECs treated with rhIGF2 in the presence or absence of rhGFBP5 ( n = 5 in each group). (G) Representative images and quantification of wound healing assay of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (H) Representative images and quantification of wound healing assay of HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (I) Representative images and quantification of transwell assay of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5 ( n = 5 in each group). (J) Representative images and quantification of transwell assay of HUVECs treated with rhIGF2 in the presence or absence of hIGFBP5 ( n = 5 in each group). (K) Representative images of Western blotting assay‐detected time‐course of p‐IGF1R, IGF1R, p‐Erk1/2, Erk1/2, p‐Ak and Akt expression of HUVECs treated with rhIGF1 in the presence or absence of rhIGFBP5. (L) Representative images of Western blotting assay‐detected time‐course of p‐IGF1R, IGF1R, p‐Erk1/2, Erk1/2, p‐Ak and Akt expression of HUVECs treated with rhIGF2 in the presence or absence of rhIGFBP5.

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Recombinant, Binding Assay, Migration, Immunofluorescence, Staining, Flow Cytometry, Wound Healing Assay, Transwell Assay, Western Blot, Expressing

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Insulin‐like growth factor‐binding protein 5 (IGFBP5) deficiency promotes glycolysis through IGF1R. (A) OCR and (B) ECAR profiles in sh‐NC‐ and sh‐IGFBP5‐infected human umbilical vein endothelial cells (HUVECs) ( n = 8 in each group). (C) Quantification of ATP production in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs. (D) OCR and (E) ECAR profiles showing the glycolytic function in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 8 in each group). (F) Quantification of glycolytic function parameters in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 8 in each group). (G) Western blotting images and (H) quantification of the expression of lactate dehydrogenase (LDH), PKM1 and PKM2 in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs. (I) OCR, (J) ECAR, (K) proton efflux rate (PER) and (L) glycoPER profiles showing glycolytic function in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of IGF1R inhibitor picropodophyllin (PPP) ( n = 6 in each group). (M) Quantification of glycolytic function parameters in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of PPP ( n = 8 in each group). (N) Western blot images and (O) quantification of the expression of LDH, PKM1 and PKM2 in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of IGF1R inhibitor PPP ( n = 6 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Infection, Western Blot, Expressing

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Deletion of insulin‐like growth factor‐binding protein 5 (IGFBP5) increases HIF1α expression by suppressing ubiquitination via ubiquitin ligase Von Hippel‐Lindau (VHL). (A) Western blotting images and quantification of HIF1α in sh‐NC‐ and sh‐IGFBP5‐infected human umbilical vein endothelial cells (HUVECs) ( n = 6 in each group). (B) Western blotting images and quantification of HIF1α in NC‐overexpression (OE‐NC)‐ and IGFBP5‐overexpression (OE‐IGFBP5)‐transfected HUVECs ( n = 6 in each group). (C) The stability of HIF1α expression in sh‐NC‐ and sh‐IGFBP5‐transfected HUVECs in the presence of 20 μg/mL cycloheximide (CHX) determined by Western blot ( n = 5 in each group). (D) The stability of HIF1α expression in OE‐NC‐ and OE‐IGFBP5‐transfected HUVECs in the presence of MG132 (MG) determined by Western blot. (E) Western blotting images of total ubiquitin, K48 ubiquitin and K63 ubiquitin of HIF1α in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 3). (F) Western blotting images and quantification of VHL in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 5 in each group). (G) Co‐immunoprecipitation of the interaction between VHL and HIF1α detected by Western blot in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs ( n = 3). (H) Western blot images and quantification of HIF1α expression in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of IGF1R inhibitor picropodophyllin (PPP) ( n = 5 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Expressing, Ubiquitin Proteomics, Western Blot, Infection, Over Expression, Transfection, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Endothelial cell (EC)‐specific deletion of IGF1R abolished IGFBP5 EKO ‐attenuated limb ischaemia. (A) Representative images detected by laser Doppler‐based tissue perfusion system in IGFBP5 EKO mice, which were infected with AAV2/9‐sh‐NC or AAV‐sh‐IGF1R after ischaemia induction (0 W) and 4 weeks after ischaemia (4 W). (B) Quantification of blood flow in the hindlimb of IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV‐sh‐IGF1R before (−1 D) and after ischaemia induction at 0 days, and 1, 2, 3 and 4 weeks ( n = 8 in each group). (C) Percentage of limb salvage, foot necrosis and limb loss in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 8 in each group). (D) Representative images of immunofluorescence staining, and quantification of CD31 (red) and DAPI (blue) in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group). (E) Representative images of haematoxylin‒eosin and picrosirius red staining, and quantification of fibrosis area in gastrocnemius of the hindlimb in ischaemic or sham in IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group). (F) Representative immunofluorescence staining images and quantification of dihydroethidium (DHE) staining for reactive oxygen species (ROS) detection in gastrocnemius of the hindlimb in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group). (G) Representative immunofluorescence staining images and quantification of TUNEL staining in gastrocnemius of the hindlimb in sham or ischaemic IGFBP5 EKO mice infected with AAV2/9‐sh‐NC or AAV2/9‐sh‐IGF1R 4 weeks after ischaemia induction ( n = 5 in each group).

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Infection, Immunofluorescence, Staining, TUNEL Assay

Journal: Clinical and Translational Medicine

Article Title: Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis

doi: 10.1002/ctm2.1725

Figure Lengend Snippet: Schematic illustration describing the underlying mechanism of insulin‐like growth factor‐binding protein 5 (IGFBP5)‐regulated endothelial cell angiogenesis post‐hindlimb ischaemia (HLI). After HLI, IGFBP5 production was promoted, which inhibited the action of IGF1/2 and the phosphorylation of IGF1R and ATP production, thereby promoting the ubiquitin‐ligase Von Hippel‐Lindau (VHL) and the ubiquitination and degradation of HIF1α. When deficiency of IGFBP5 in endothelial cells (ECs), the IGF1R phosphorylation was promoted, the ATP production was promoted, and the ubiquitination of HIF1α was suppressed, which promoted angiogenesis post‐HLI.

Article Snippet: For siRNA transfection, HUVECs were transfected with RNAi MAX and IGFBP5‐siRNA or control siRNA for 48 h. Human IGFBP5 recombinant protein was added to HUVECs at 200 ng/mL concentration for 72 h. To investigate the effect of IGF1 (Cat#:8917SC, Cell Signalling Technology) or IGF2 (Cat#: 292‐G2‐050, R&D systems), HUVECs were treated with IGF1 (100 ng/mL) or IGF2 (100 ng/mL) and then incubated with human IGFBP5 recombinant protein (100 ng/mL) or vehicle in serum‐free ECM.

Techniques: Binding Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: Immunity

Article Title: Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut.

doi: 10.1016/j.immuni.2008.05.014

Figure Lengend Snippet: Figure 6. RORgt+LTi Cells and Gut Mf-DCs Support Class Switching and Differentiation to IgA Plasma Cells through SCs (A and B) IgA-depleted spleen cells were cultured for 5 days without (A) and with (B) conditioned media of 4-day culture supernatant of SCs, in the presence of stimulants and antibodies indicated. The GFP+RORgt+LTi cells in (A) appear as IgA intermediate B220 population (FITC-IgA; APC-B220). (C) Total amount of IgA in 5-day cultures of IgA-depleted spleen cells in conditioned media (1), conditioned media with TGF-b antibodies (2) or control antibodies (3), and conditioned media with TACI-Ig and BCMA-Ig (4). (D) Total amount of TGF-b1 in culture supernatants shown in (B), as determined by ELISA. (E) Real-time RT-PCR analyses of the indicated MMPs, BAFF, and APRIL, expressed as relative amounts of mRNA normalized to GAPDH. Mean ± standard deviation of three independent experiments. (F) IgA-depleted spleen cells were cultured for 5 days in the presence of LPS and APRIL and BAFF, without or with IL5 and TGF-b1. Percentages of B220+IgA+

Article Snippet: IgA-depleted spleen cells were cultured in D-MEM medium supplemented with 10% fetal calf serum (FCS), L-glutamine, sodium pyruvate, HEPES, and 2-mercaptoethanol, without or with 50% of conditioned media (from a 4-day culture supernatant of gut SCs from C57BL/6 mice), in the presence of LPS (20 mg/ml; Sigma), pan-specific TGF-b antibodies (35 mg/ml), control IgG (35 mg/ml), TACI-Ig and BCMA-Ig (30 mg/ml), recombinant mouse IL5 (100U/ml), human TGF-b1 (1 ng/ml), all from R&D System for 5 days.

Techniques: Clinical Proteomics, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Standard Deviation