Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: After exposure of HCFs to various concentrations of zymosan, the expression of Cx43 was examined by Western blot analysis; B: The immunoblots were subjected to densitometric analysis in order to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's multiple comparison test) versus the control (no zymosan).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Comparison, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: After the exposure of HCFs to zymosan (600 µg/mL) for the indicated time, the expression of Cx43 was examined by Western blot analysis; B: The immunoblots were subjected to densitometric analysis in order to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's multiple comparison test) versus the control (no zymosan).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Comparison, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: After HCFs were incubated with or without zymosan (600 µg/mL) for 24h, the Cx43 mRNA level in the cells was then determined by qRT-PCR analysis. The error bars represent the standard deviation. aP<0.05 (Student's t-test) versus the control (no zymosan).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Incubation, Quantitative RT-PCR, Standard Deviation, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: After HCFs were treated with PD98059, SB203580, or JNK inhibitor II (at 1, 3, and 10 µmol/L), respectively, in the absence or presence of zymosan (600 µg/mL), the expression of Cx43 was then examined by Western blot analysis; B: Densitometric analysis was performed for the immunoblots to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's test) versus the control (no zymosan); bP<0.05 (Dunnett's test) versus the corresponding control (zymosan only).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: A: HCFs were treated with IKK2 inhibitor IV (1, 3, and 10 µmol/L) in the absence or presence of zymosan (600 µg/mL). The expression of Cx43 was then examined by Western blot analysis. B: The immunoblots were subjected to densitometric analysis in order to determine the band intensity. The error bars represent the standard deviation. aP<0.05 (Dunnett's test) versus the control (no zymosan). bP<0.05 (Dunnett's test) versus the corresponding control (zymosan only).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: International Journal of Ophthalmology

Article Title: Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

doi: 10.18240/ijo.2021.03.02

Figure Lengend Snippet: HCFs were treated with the ERK inhibitor PD98059, JNK inhibitor II, or IKK2 inhibitor IV (10 µmol/L), respectively, in the absence or presence of zymosan (600 µg/mL). A scrape-loading assay with Lucifer yellow was used to measure the GJIC activity. A: Representative fluorescence microscopy images of fixed cells from different treatments (scale bar, 100 µm); B: The maximum distances from the scrape to dye fluorescence were quantified to show the GJIC activity. The error bars represent the standard deviation. aP<0.05 (Dunnett's test) versus the control (no zymosan). bP<0.05 (Dunnett's test) versus the corresponding control (zymosan only).

Article Snippet: Culture of Human Corneal Fibroblasts HCFs were purchased from ScienCell Research Laboratories (cat. no. #6520; Carlsbad, CA, USA) and cultured in MEM containing 10% FBS (5% CO 2 , 37°C).

Techniques: Activity Assay, Fluorescence, Microscopy, Standard Deviation, Control

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: Primary HCFs (6330, Lot number 9580, ScienCell) were isolated from the healthy male heart.

Techniques: Activation Assay, Phospho-proteomics, 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: Primary HCFs (6330, Lot number 9580, ScienCell) were isolated from the healthy male heart.

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: Primary HCFs (6330, Lot number 9580, ScienCell) were isolated from the healthy male heart.

Techniques: Immunofluorescence, Western Blot, Activation Assay, Comparison, 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: IL11 promotes the translation of proline-rich pro-fibrotic genes, including itself, through EPRS activity. (A) Amino acid sequence of human IL11, proline residues highlighted in yellow. (B) IL11 levels in the supernatant following TGFβ1 or HyperIL11 stimulation in the presence of Halofuginone (Halo) or Cycloheximide (CHX) ( n = 3). (C,D) (C) Quantification ( n = 14) and (D) immunofluorescence images (scale bars, 100 μm; n = 3)) of Halofuginone-treated TGFβ1/IL11-stimulated HCFs for Collagen I staining. (E,F) (E) EPRS mRNA ( n = 3) and (F) EPRS protein ( n = 2) expression levels after knockdown using siEPRS. (G–I) (G) IL11 levels in the supernatant ( n = 4), (H) quantification (n = 14), and (I) immunofluorescence images (scale bars, 100 μm; n = 3) of Collagen I in stimulated HCFs subjected to siEPRS. (B–I) primary HCFs; IL11/TGFβ1/HyperIL11 (10 ng/ml), Cycloheximide (CHX, 100 μg/ml), Halofuginione (100 nM), non-targeting siRNA (siNT)/EPRS siRNA (siEPRS) (12.5 nM); 24 h. (B,C, G,H) 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; (E) Data are shown as mean ± SD, 2-tailed t -test. Signaling relationships between nintedanib, pirfenidone and anti-IL11.

Article Snippet: Primary HCFs (6330, Lot number 9580, ScienCell) were isolated from the healthy male heart.

Techniques: Activity Assay, Sequencing, Immunofluorescence, Staining, Expressing, Knockdown, 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: Nintedanib, Pirfenidone and anti-IL11 have different anti-fibrotic mechanisms of action in cardiac fibroblasts. (A) Western blots showing activation status of ERK, STAT3, mTOR, p70S6K (T389), S6RP, and Caspase3, and protein expression of ⍺SMA, BIP, XBP1-S, and CHOP following treatment with nintedanib, pirfenidone, IgG, anti-IL11, a combination of nintedanib, anti-IL11, U0126 or S3I-201 from TGFβ1-stimulated HCFs ( n = 1). (B) Western blots of p-ERK, ERK, p-STAT3, STAT3, ⍺SMA, BIP, CHOP, Cleaved Caspase 3, Caspase 3, and GAPDH from HCFs treated with different concentrations of nintedanib in the absence or presence of TGFβ1 ( n = 1). (C) Western blots of p-ERK, ERK, p-STAT3, STAT3, ⍺SMA, BIP, CHOP, Cleaved Caspase 3, Caspase 3, XBP1-S, and GAPDH from PDGF-stimulated HCFs ( n = 1). (D) Western blots of p-PDGFRB, PDGFRB, p-ERK, ERK, p-STAT3, STAT3, BIP, CHOP, Cleaved Caspase 3, Caspase 3, and GAPDH from PDGF-stimulated HCFs treated with different concentrations of nintedanib ( n = 1). (E) Densitometry analysis of ⍺SMA versus BIP expression from TGFβ1-stimulated HCFs treated with different concentrations of nintedanib ( n = 1). (F) Schematic overview and timeline of key pro-fibrotic events that occur downstream of TGFβ1-stimulation in human fibroblasts. (A–E) primary HCFs, 24 h; TGFβ1 (10 ng/ml), PDGF (20 ng/ml), IgG/anti-IL11 (2 μg/ml), nintedanib (2 µM), pirfenidone (0.3 mg/ml), U0126 (10 µM), S3I-201 (20 µM), unless otherwise specified.

Article Snippet: Primary HCFs (6330, Lot number 9580, ScienCell) were isolated from the healthy male heart.

Techniques: Western Blot, Activation Assay, Expressing

Journal: Molecular Medicine Reports

Article Title: P21-activated kinase 1 mediates angiotensin II-induced differentiation of human atrial fibroblasts via the JNK/c-Jun pathway

doi: 10.3892/mmr.2021.11846

Figure Lengend Snippet: PAK1 inhibition with IPA-3 attenuates AngII-induced migration of HCFs. (A) Representative images of wound healing assays showing that IPA-3 (5 µM) attenuated AngII-induced migration ability of HCFs (scale bar, 200 µm). (B) Wound closure rate as % of wound area at 24 h vs. original wound area (0 h). (C) Representative images from Transwell migration assays showing that treatment of HCFs with 5 µM IPA-3 decreased AngII-induced cell migration ability (scale bar, 200 µm). (D) Cell migration rate in the different groups (%). Data shown are mean fold change ± SEM. *P<0.05, **P<0.01 vs. control; ## P<0.01 vs. AngII. PAK1, p21-activated kinase 1; AngII, angiotensin II; HCFs, human cardiac fibroblasts.

Article Snippet: Adult HCFs and fibroblast medium-2 were purchased from ScienCell Research Laboratories, Inc. (cat. no. 6320; sciencellonline.com/human-cardiac-fibroblasts-juvenile-atrial.html ).

Techniques: Inhibition, Migration, Control

Journal: Molecular Medicine Reports

Article Title: P21-activated kinase 1 mediates angiotensin II-induced differentiation of human atrial fibroblasts via the JNK/c-Jun pathway

doi: 10.3892/mmr.2021.11846

Figure Lengend Snippet: PAK1 knockdown alleviates AngII-induced migration of HCFs. (A) Representative images of wound healing assays showing that PAK1 knockdown with PAK1-shRNA attenuated AngII-induced migration of HCFs (scale bar, 200 µm). (B) Wound closure rate as % of wound area at 24 h vs. original wound area (0 h). (C) Representative images from Transwell migration assays showing that treatment of HCFs with PAK1-shRNA decreased AngII-induced cell migration ability (scale bar, 200 µm). (D) Cell migration rate in different groups. Data are presented as the mean fold change ± SEM. *P<0.05, **P<0.01 vs. control; ## P<0.01 vs. Ang II. PAK1, p21-activated kinase 1; AngII, angiotensin II; HCFs, human cardiac fibroblasts; sh, short hairpin; NC, negative control.

Article Snippet: Adult HCFs and fibroblast medium-2 were purchased from ScienCell Research Laboratories, Inc. (cat. no. 6320; sciencellonline.com/human-cardiac-fibroblasts-juvenile-atrial.html ).

Techniques: Knockdown, Migration, shRNA, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: P21-activated kinase 1 mediates angiotensin II-induced differentiation of human atrial fibroblasts via the JNK/c-Jun pathway

doi: 10.3892/mmr.2021.11846

Figure Lengend Snippet: IPA-3 inhibits AngII-induced transdifferentiation and proliferation of HCFs. (A) Representative images of western blot assays and (B and C) histograms, showing that IPA-3 inhibits AngII-mediated upregulation of (B) collagen I and (C) α-SMA. (D) Representative images of western blot assays and (E) histogram showing that AngII increased the expression levels of PAK1 in HCFs. (F) Cell proliferation measured by Cell Counting Kit-8 assay showing that IPA-3 inhibited AngII-induced cell proliferation. Data are presented as the mean fold change ± SEM. *P<0.05, **P<0.01 vs. control; ## P<0.01 vs. AngII. AngII, angiotensin II; HCFs, human cardiac fibroblasts; α-SMA, α smooth muscle actin; PAK1, p21-activated kinase 1.

Article Snippet: Adult HCFs and fibroblast medium-2 were purchased from ScienCell Research Laboratories, Inc. (cat. no. 6320; sciencellonline.com/human-cardiac-fibroblasts-juvenile-atrial.html ).

Techniques: Western Blot, Expressing, Cell Counting, Control

Journal: Molecular Medicine Reports

Article Title: P21-activated kinase 1 mediates angiotensin II-induced differentiation of human atrial fibroblasts via the JNK/c-Jun pathway

doi: 10.3892/mmr.2021.11846

Figure Lengend Snippet: PAK1 knockdown inhibits Ang II-induced transdifferentiation of HCFs. (A) PAK-shRNA decreased mRNA expression levels of PAK1. (B and C) Western blot and histogram showing that PAK1 knockdown decreased the protein expression levels of PAK1. (D) Representative images of western blot assays and (E and F) histograms are presented, showing that PAK1-knockdown inhibited AngII-mediated upregulation of (E) α-SMA and (F) collagen I. Data are presented as the mean fold change ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. NC; ## P<0.01 vs. AngII. PAK1, p21-activated kinase 1; AngII, angiotensin II; HCFs, human cardiac fibroblasts; sh, short hairpin; α-SMA, α smooth muscle actin; NC, negative control.

Article Snippet: Adult HCFs and fibroblast medium-2 were purchased from ScienCell Research Laboratories, Inc. (cat. no. 6320; sciencellonline.com/human-cardiac-fibroblasts-juvenile-atrial.html ).

Techniques: Knockdown, shRNA, Expressing, Western Blot, Negative Control

Journal: Molecular Medicine Reports

Article Title: P21-activated kinase 1 mediates angiotensin II-induced differentiation of human atrial fibroblasts via the JNK/c-Jun pathway

doi: 10.3892/mmr.2021.11846

Figure Lengend Snippet: JNK/c-Jun signaling pathway mediates the effects of PAK1 in HCFs. (A) Representative images of western blot assays and histograms showing that transduction of HCFs with PAK1-shRNA attenuated AngII-induced phosphorylation of (B) JNK and (C) c-Jun. *P<0.05, **P<0.01 vs. NC. ## P<0.01 vs. AngII. (D) Representative images of western blot assays and histograms showing that PAK1 inhibition with IPA-3 (5 µM) attenuated AngII-induced phosphorylation of (E) JNK and (F) c-Jun. *P<0.05, **P<0.01 vs. control. ## P<0.01 vs. AngII. (G) Mechanism underlying the effect of PAK1 on the migration and transdifferentiation of HCFs. Data are presented as the mean fold change ± SEM. JNK, Janus kinase; PAK1, p21-activated kinase 1; HCF, human cardiac fibroblast; sh, short hairpin; AngII, angiotensin II; NC, negative control; p, phosphorylated; α-SMA, α smooth muscle actin.

Article Snippet: Adult HCFs and fibroblast medium-2 were purchased from ScienCell Research Laboratories, Inc. (cat. no. 6320; sciencellonline.com/human-cardiac-fibroblasts-juvenile-atrial.html ).

Techniques: Western Blot, Transduction, shRNA, Phospho-proteomics, Inhibition, Control, Migration, Negative Control