Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 messenger ribonucleic acid (mRNA) (detected with reverse transcription polymerase chain reaction (RTPCR)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas plus immunoglobulin G (IgG) isotype antibody treated controls. B. IL-11 protein (detected with enzyme-linked immunosorbent assay (ELISA)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in human proximal tubule cells.

Article Snippet: Immunohistochemistry detected mouse kidney IL-11 protein expression and localization 4 hr after anesthesia under pentobarbital or 1.2% isoflurane with rat anti-IL-11 antibody (MAB418, 1:50 dilution; R&D Systems) and biotin conjugated anti-rat IgG (1:100 dilution, Vector Laboratories, Burlingame, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 h (N = 4). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas and immunoglobulin G (IgG) isotype antibody treated controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was also quantified to normalize lane loading. * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in mouse proximal tubule cells.

Article Snippet: Immunohistochemistry detected mouse kidney IL-11 protein expression and localization 4 hr after anesthesia under pentobarbital or 1.2% isoflurane with rat anti-IL-11 antibody (MAB418, 1:50 dilution; R&D Systems) and biotin conjugated anti-rat IgG (1:100 dilution, Vector Laboratories, Burlingame, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Naïve IL-11 wild-type (WT) mice were exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h. A. Representative bands for IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in mouse kidney (N = 4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal loading control. B. Kidney lysate IL-11 protein (detected by enzyme-linked immunosorbent assay (ELISA)) in IL-11 naïve WT mice exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h (N = 4). To neutralize TGF-β1 in vivo, some IL-11 WT mice were injected with 5 mg/kg monoclonal anti-TGF-β1 (MAB240) antibody intravenous injection. TGF-β1 neutralization prevented the induction of IL-11 after isoflurane anesthesia. * P < 0.05 vs. pentobarbital anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with IgG isotype antibody. Error bars represent 1 SD. Isoflurane anesthesia significantly increased kidney IL-11 mRNA and protein expression in mice.

Article Snippet: Immunohistochemistry detected mouse kidney IL-11 protein expression and localization 4 hr after anesthesia under pentobarbital or 1.2% isoflurane with rat anti-IL-11 antibody (MAB418, 1:50 dilution; R&D Systems) and biotin conjugated anti-rat IgG (1:100 dilution, Vector Laboratories, Burlingame, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, In Vivo, Injection, Neutralization

Journal: Anesthesiology

Article Title: Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 immunohistochemistry (400× shown, N = 4) in kidneys from IL-11 receptor wild type (IL-11R WT) mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. IL-11 staining was darker in kidneys of mice anesthetized with isoflurane. TGF-β1 neutralizing antibody attenuated isoflurane-mediated increase in IL-11 immunoreactivity. Finally, IL-11 staining was not visible in kidneys from mice stained with negative isotype control antibodies (representative of four experiments). B. Quantifications of renal tubular IL-11 staining in mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. Kidney IL-11 immunoreactivity significantly increased in mice anesthetized with isoflurane and attenuated with TGF-β1 neutralizing antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. * P < 0.05 vs. pentobarbital-anesthetized mice. Error bars represent 1 SD.

Article Snippet: Immunohistochemistry detected mouse kidney IL-11 protein expression and localization 4 hr after anesthesia under pentobarbital or 1.2% isoflurane with rat anti-IL-11 antibody (MAB418, 1:50 dilution; R&D Systems) and biotin conjugated anti-rat IgG (1:100 dilution, Vector Laboratories, Burlingame, CA).

Techniques: Immunohistochemistry, Staining

Journal: Frontiers in Molecular Biosciences

Article Title: Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts

doi: 10.3389/fmolb.2021.740650

Figure Lengend Snippet: TGFβ1-/IL11-driven fibrogenesis is coincident with activation of ERK, unrelated to STAT3 phosphorylation and shows species-specific effects. (A) Western blots of ERK and STAT3 activation in TGFβ1-stimulated HCFs over a time course ( n = 1). (B) Representative immunofluorescence (IF) images of IL11RA, and gp130 expression in HCFs (scale bars, 100 μm; n = 3). (C,D) (C) Western blot analysis of p-ERK, ERK, p-STAT3, STAT3, and ⍺SMA ( n = 1) and (D) IF images of Collagen I staining ( n = 3) in TGFβ1-stimulated HCFs in the presence of either IgG, anti-IL11, or anti-IL11RA. (E) Western blots of ERK and STAT3 activation status in IL11-stimulated HCFs over a time course ( n = 1). (F) Western blots showing ERK and STAT3 activation status in HCFs following stimulation with low and high dose IL11 ( n = 1). (G–I) Effects of anti-IL11, anti-IL11RA, or anti-gp130 on inhibiting (G–H) ERK and STAT3 activation ( n = 1) and (I) Collagen I Induction ( n = 3) in IL11-stimulated HCFs. (J) IF images (scale bars, 100 µm) of IL11RA and gp130 in MCFs isolated from wild-type (WT) and Il11ra1 −/− mice ( n = 3). (K–M) Dose-dependent effects of rmIL11 and rhIL11 on (K) ERK and STAT3 activation status in wild-type (WT) MCFs, on (L) STAT3 activation (15 m) and (M) ERK activation and ⍺SMA expression (24 h) in WT and Il11ra1 −/− MCFs ( n = 1). (N) Schematic showing the effects of rmIL11 or rhIL11 on signaling and fibroblast activation in mouse fibroblasts. (A–I) primary HCFs; 24 h, (J–M) primary MCFs, (A–M) IL11/TGFβ1 (10 ng/ml), unless otherwise specified. BL: Baseline. Inhibition of STAT3 activity results in ER stress causing fibroblast dysfunction and death.

Article Snippet: BIP (3177, CST), Cleaved-Caspase 3 (9664, CST), Caspase 3 (9662, CST), CHOP (5554, CST), Collagen I (ab34710, Abcam), phospho-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), neutralizing anti-human gp130 (MAB628, R&D Systems), anti-gp130 (PA5-99526, Thermo Fisher), gp130 (PA5-28932, Thermo Fisher, IF), IgG (11E10, Aldevron), neutralizing anti-IL11 (X203, Aldevron), commercial anti-IL11 (MAB218, R&D Systems), neutralizing anti-IL11RA (X209, Aldevron), IL11RA (ab125015, abcam, IF), phospho-mTOR (2971, CST), mTOR (2972,CST), phospho-p70S6K (9205, CST), p70S6K (2708, CST), p-PDGFRB (3124, CST), PDGFRB (3169, CST), phospho-S6 ribosomal protein (4858, CST), S6 ribosomal protein (2217, CST), ⍺-SMA (ab7817, Abcam, Operetta), ⍺-SMA (19245, CST, WB), phospho-SMAD2 (3108, CST), SMAD2 (5339, CST), phospho-STAT3 (4113, CST), STAT3 (4904, CST), XBP-1 (sc-8015, SantaCruz), mouse Alexa Fluor 488 secondary antibody (ab150113, Abcam), mouse HRP (7076, CST), rabbit Alexa Fluor 488 secondary antibody (ab150077, Abcam), rabbit HRP (7074, CST).

Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing, Staining, Isolation, Inhibition, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts

doi: 10.3389/fmolb.2021.740650

Figure Lengend Snippet: Inhibition of STAT3 in TGFβ1-or IL11-stimulated fibroblasts causes ER stress-related fibroblast dysfunction mimicking effects of generic ER stressors. (A–E) Effects of U0126 or S3I-201 on rhIL11-stimulated HCFs. (A,B) IF images (scale bars, 100 μm; n = 3) and (C,D) quantification of ⍺SMA +ve cells and Collagen I immunostaining ( n = 14). (E) Western blots showing ERK, STAT3, and Caspase3 activation status and ⍺SMA and CHOP protein expression ( n = 1). (F–I) Quantification of (F,G) ⍺SMA +ve cells and (H,I) Collagen I immunostaining following stimulation with (F, H) rhIL11 or (G, I) TGFβ1 in the presence of Stattic ( n = 14). (J) Effects of S3I-201 or Stattic on the activation of ERK, STAT3, and Caspase3 and on the expression of ⍺SMA, CHOP, and XBP1-S at baseline and in TGFFβ1-or IL11-stimulated HCFs ( n = 1). (K) Western blots of BIP and XBP1-S from IgG/anti-IL11/anti-IL11RA-treated TGFβ-stimulated HCFs ( n = 1). (L,M) (L) Representative IF images (scale bars, 100 µm) of ⍺SMA +ve cells and Collagen I ( n = 3) and (M) Western blot analysis of pERK, ERK, pSTAT3, STAT3, ⍺SMA, BIP, XBP1-S, CHOP, Cleaved Caspase3, Caspase3, and GAPDH from TGFβ1-or rhIL11-stimulated HCFs ( n = 1). (A–M) primary HCFs; 24 h; rhIL11/TGFβ1 (10 ng/ml), U0126 (10 µM), S3I-201 (20 µM), Stattic (2.5 µM), IgG/anti-IL11/anti-IL11RA (2 μg/ml), Tunicamycin (5 μg/ml), Thapsigargin (300 nM). (C,D, F–I) Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers); one-way ANOVA with Tukey’s correction. BL: Baseline. TGFβ1-stimulated protein synthesis is IL11-/ERK- and mTOR-dependent.

Article Snippet: BIP (3177, CST), Cleaved-Caspase 3 (9664, CST), Caspase 3 (9662, CST), CHOP (5554, CST), Collagen I (ab34710, Abcam), phospho-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), neutralizing anti-human gp130 (MAB628, R&D Systems), anti-gp130 (PA5-99526, Thermo Fisher), gp130 (PA5-28932, Thermo Fisher, IF), IgG (11E10, Aldevron), neutralizing anti-IL11 (X203, Aldevron), commercial anti-IL11 (MAB218, R&D Systems), neutralizing anti-IL11RA (X209, Aldevron), IL11RA (ab125015, abcam, IF), phospho-mTOR (2971, CST), mTOR (2972,CST), phospho-p70S6K (9205, CST), p70S6K (2708, CST), p-PDGFRB (3124, CST), PDGFRB (3169, CST), phospho-S6 ribosomal protein (4858, CST), S6 ribosomal protein (2217, CST), ⍺-SMA (ab7817, Abcam, Operetta), ⍺-SMA (19245, CST, WB), phospho-SMAD2 (3108, CST), SMAD2 (5339, CST), phospho-STAT3 (4113, CST), STAT3 (4904, CST), XBP-1 (sc-8015, SantaCruz), mouse Alexa Fluor 488 secondary antibody (ab150113, Abcam), mouse HRP (7076, CST), rabbit Alexa Fluor 488 secondary antibody (ab150077, Abcam), rabbit HRP (7074, CST).

Techniques: Inhibition, Immunostaining, Western Blot, Activation Assay, Expressing, Whisker Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts

doi: 10.3389/fmolb.2021.740650

Figure Lengend Snippet: TGFβ1-and IL11-stimulated protein synthesis is ERK- and mTOR-dependent. (A) Schematic for experiments shown in (B–D) . (B–D) (B,C) Immunofluorescence images (scale bars, 100 μm; n = 3) and (D) quantification of Alexa Fluor™ 488 - OPP signal in HCFs ( n = 14) following treatments shown in (A) . (E–G) Western blots of p-p70S6K (T389), p70S6K, p-S6RP, S6RP from (E) TGFβ1 or (F) rhIL11-stimulated HCFs over a time course and on (G) IgG, anti-IL11, or anti-IL11RA-treated TGFβ1-stimulated HCFs ( n = 1). (H) Effects of U0126 on TGFβ1 or rhIL11-induced ERK, p70S6K (T389), and S6RP activation ( n = 1). (I) Comparison effects of Rapamycin, Wortmannin, and U0126 on rhIL11-induced ERK, mTOR, p70S6K, and S6RP activation ( n = 1). (B–I) primary HCFs; IL11/TGFβ1 (10 ng/ml), OPP (20 µM), CHX (100 μg/ml), IgG/anti-IL11/anti-IL11RA (2 μg/ml), U0126 (10 µM), Rapamycin (10 nM), Wortmannin (1 µM); (B–D) 8 h, (G–I) 24 h. (D) Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers); one-way ANOVA with Tukey’s correction. IL11 stimulates translation of proline-rich pro-fibrotic genes via EPRS.

Article Snippet: BIP (3177, CST), Cleaved-Caspase 3 (9664, CST), Caspase 3 (9662, CST), CHOP (5554, CST), Collagen I (ab34710, Abcam), phospho-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), neutralizing anti-human gp130 (MAB628, R&D Systems), anti-gp130 (PA5-99526, Thermo Fisher), gp130 (PA5-28932, Thermo Fisher, IF), IgG (11E10, Aldevron), neutralizing anti-IL11 (X203, Aldevron), commercial anti-IL11 (MAB218, R&D Systems), neutralizing anti-IL11RA (X209, Aldevron), IL11RA (ab125015, abcam, IF), phospho-mTOR (2971, CST), mTOR (2972,CST), phospho-p70S6K (9205, CST), p70S6K (2708, CST), p-PDGFRB (3124, CST), PDGFRB (3169, CST), phospho-S6 ribosomal protein (4858, CST), S6 ribosomal protein (2217, CST), ⍺-SMA (ab7817, Abcam, Operetta), ⍺-SMA (19245, CST, WB), phospho-SMAD2 (3108, CST), SMAD2 (5339, CST), phospho-STAT3 (4113, CST), STAT3 (4904, CST), XBP-1 (sc-8015, SantaCruz), mouse Alexa Fluor 488 secondary antibody (ab150113, Abcam), mouse HRP (7076, CST), rabbit Alexa Fluor 488 secondary antibody (ab150077, Abcam), rabbit HRP (7074, CST).

Techniques: Immunofluorescence, Western Blot, Activation Assay, Whisker Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts

doi: 10.3389/fmolb.2021.740650

Figure Lengend Snippet: TGFβ1-and IL11-stimulated protein synthesis is ERK- and mTOR-dependent. (A) Schematic for experiments shown in (B–D) . (B–D) (B,C) Immunofluorescence images (scale bars, 100 μm; n = 3) and (D) quantification of Alexa Fluor™ 488 - OPP signal in HCFs ( n = 14) following treatments shown in (A) . (E–G) Western blots of p-p70S6K (T389), p70S6K, p-S6RP, S6RP from (E) TGFβ1 or (F) rhIL11-stimulated HCFs over a time course and on (G) IgG, anti-IL11, or anti-IL11RA-treated TGFβ1-stimulated HCFs ( n = 1). (H) Effects of U0126 on TGFβ1 or rhIL11-induced ERK, p70S6K (T389), and S6RP activation ( n = 1). (I) Comparison effects of Rapamycin, Wortmannin, and U0126 on rhIL11-induced ERK, mTOR, p70S6K, and S6RP activation ( n = 1). (B–I) primary HCFs; IL11/TGFβ1 (10 ng/ml), OPP (20 µM), CHX (100 μg/ml), IgG/anti-IL11/anti-IL11RA (2 μg/ml), U0126 (10 µM), Rapamycin (10 nM), Wortmannin (1 µM); (B–D) 8 h, (G–I) 24 h. (D) Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers); one-way ANOVA with Tukey’s correction. IL11 stimulates translation of proline-rich pro-fibrotic genes via EPRS.

Article Snippet: BIP (3177, CST), Cleaved-Caspase 3 (9664, CST), Caspase 3 (9662, CST), CHOP (5554, CST), Collagen I (ab34710, Abcam), phospho-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), neutralizing anti-human gp130 (MAB628, R&D Systems), anti-gp130 (PA5-99526, Thermo Fisher), gp130 (PA5-28932, Thermo Fisher, IF), IgG (11E10, Aldevron), neutralizing anti-IL11 (X203, Aldevron), commercial anti-IL11 (MAB218, R&D Systems), neutralizing anti-IL11RA (X209, Aldevron), IL11RA (ab125015, abcam, IF), phospho-mTOR (2971, CST), mTOR (2972,CST), phospho-p70S6K (9205, CST), p70S6K (2708, CST), p-PDGFRB (3124, CST), PDGFRB (3169, CST), phospho-S6 ribosomal protein (4858, CST), S6 ribosomal protein (2217, CST), ⍺-SMA (ab7817, Abcam, Operetta), ⍺-SMA (19245, CST, WB), phospho-SMAD2 (3108, CST), SMAD2 (5339, CST), phospho-STAT3 (4113, CST), STAT3 (4904, CST), XBP-1 (sc-8015, SantaCruz), mouse Alexa Fluor 488 secondary antibody (ab150113, Abcam), mouse HRP (7076, CST), rabbit Alexa Fluor 488 secondary antibody (ab150077, Abcam), rabbit HRP (7074, CST).

Techniques: Immunofluorescence, Western Blot, Activation Assay, Whisker Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts

doi: 10.3389/fmolb.2021.740650

Figure Lengend Snippet: TGFβ1-and IL11-stimulated protein synthesis is ERK- and mTOR-dependent. (A) Schematic for experiments shown in (B–D) . (B–D) (B,C) Immunofluorescence images (scale bars, 100 μm; n = 3) and (D) quantification of Alexa Fluor™ 488 - OPP signal in HCFs ( n = 14) following treatments shown in (A) . (E–G) Western blots of p-p70S6K (T389), p70S6K, p-S6RP, S6RP from (E) TGFβ1 or (F) rhIL11-stimulated HCFs over a time course and on (G) IgG, anti-IL11, or anti-IL11RA-treated TGFβ1-stimulated HCFs ( n = 1). (H) Effects of U0126 on TGFβ1 or rhIL11-induced ERK, p70S6K (T389), and S6RP activation ( n = 1). (I) Comparison effects of Rapamycin, Wortmannin, and U0126 on rhIL11-induced ERK, mTOR, p70S6K, and S6RP activation ( n = 1). (B–I) primary HCFs; IL11/TGFβ1 (10 ng/ml), OPP (20 µM), CHX (100 μg/ml), IgG/anti-IL11/anti-IL11RA (2 μg/ml), U0126 (10 µM), Rapamycin (10 nM), Wortmannin (1 µM); (B–D) 8 h, (G–I) 24 h. (D) Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers); one-way ANOVA with Tukey’s correction. IL11 stimulates translation of proline-rich pro-fibrotic genes via EPRS.

Article Snippet: BIP (3177, CST), Cleaved-Caspase 3 (9664, CST), Caspase 3 (9662, CST), CHOP (5554, CST), Collagen I (ab34710, Abcam), phospho-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), neutralizing anti-human gp130 (MAB628, R&D Systems), anti-gp130 (PA5-99526, Thermo Fisher), gp130 (PA5-28932, Thermo Fisher, IF), IgG (11E10, Aldevron), neutralizing anti-IL11 (X203, Aldevron), commercial anti-IL11 (MAB218, R&D Systems), neutralizing anti-IL11RA (X209, Aldevron), IL11RA (ab125015, abcam, IF), phospho-mTOR (2971, CST), mTOR (2972,CST), phospho-p70S6K (9205, CST), p70S6K (2708, CST), p-PDGFRB (3124, CST), PDGFRB (3169, CST), phospho-S6 ribosomal protein (4858, CST), S6 ribosomal protein (2217, CST), ⍺-SMA (ab7817, Abcam, Operetta), ⍺-SMA (19245, CST, WB), phospho-SMAD2 (3108, CST), SMAD2 (5339, CST), phospho-STAT3 (4113, CST), STAT3 (4904, CST), XBP-1 (sc-8015, SantaCruz), mouse Alexa Fluor 488 secondary antibody (ab150113, Abcam), mouse HRP (7076, CST), rabbit Alexa Fluor 488 secondary antibody (ab150077, Abcam), rabbit HRP (7074, CST).

Techniques: Immunofluorescence, Western Blot, Activation Assay, Whisker Assay