Journal: Investigative Ophthalmology & Visual Science

Article Title: Defining the Role of Mitochondrial Fission in Corneal Myofibroblast Differentiation

doi: 10.1167/iovs.63.4.2

Figure Lengend Snippet: Intracellular mediators underlying anti-fibrotic effects of Mdivi-1. ( A ) Representative Western blot depicting phospho- and total-SMAD3 expression levels in protein extracts from cultured cat corneal fibroblasts. Cells were incubated ± 10 µM Mdivi-1, ± 1 ng/mL TGF-β1 and finally 2.3 µM SB431542 with 1 ng/mL TGF-β1, as indicated. ( B ) Plot of densitometric ratios for phospho-SMAD3 expression relative to total-SMAD2/3, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 116.3, P < 0.0001. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 alone are denoted: * P < 0.05, # P < 0.01. ( C ) Representative Western blots of corneal fibroblast extracts for phosphor-p54 and -p46 and total-JNK expression under the conditions indicated. ( D ) Plot of densitometric ratios for p-p54 JNK expression relative to total-p54 JNK, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 7.48, P = 0.01884. Significances relative to the TGF-β1 condition were computed and are denoted as in B . ( E ) Representative Western blot of corneal fibroblast extracts showing phospho-p38, phospho-ERK, and phospho-AKT expression levels under the conditions indicated. ( F ) Plot of densitometry ratios for phosphorylated proteins relative to β-actin, normalized to the TGF-β1 treatment. One-way repeated measures ANOVAs demonstrated significant effects of treatments for all 3 molecules: p38 F(3,11) = 37.52, P = 0.0003; pERK F(3,11) = 11.12, P = 0.0073; pAKT F(3,11) = 8.85, P = 0.0127. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 are indicated as in B and D . For all graphs, data shown are means ± SD from 3 experimental replicates.

Article Snippet: Blots were incubated overnight at 4°C containing primary antibodies to the following targets at the dilutions indicated: Type 1 collagen (COL1; 1:2000; no. LF-68, kindly provided by Dr. Larry W. Fisher, NIH, Bethesda, MD, USA), total Fibronectin (t-FN; 1:2000; #H-300; Santa Cruz Inc.), α-SMA (1:10,000; no. MA5-11547; Thermo Fisher Scientific, Waltham, MA, USA), total SMAD 2/3 (1:2000; no. 8685; Cell Signaling Technology, Danvers, MA, USA), phosphorylated small mothers against decapentaplegic 3 (SMAD3) (S423+S425; 1:1000; no. P00059-1; Boster Technology, Pleasanton, CA, USA), phospho-p46/54 JNK Thr183/Tyr185 (1:1000; no. 4671; Cell Signaling Technology), phospho-p38 MAPK Thr180/Tyr182 (1:1000; no. 9215; Cell Signaling Technology), phospho-p44/42 MAPK Thr202/Tyr204 (1:1000; no. 9106; Cell Signaling Technology), phospho-AKT Ser473 (1:1000; no. 4060; Cell Signaling Technology), total-p46/54 JNK (1:1000; no. 9252; Cell Signaling Technology), p-Drp1 Ser616 (1:500; no. 3455; Cell Signaling Technology), total-Drp1 (1:1000; no. 611112; BD Biosciences, San Jose, CA, USA), Opa1 (1:1000; no. 612606; BD Biosciences), Mfn1 (1:1000; ab57602; Abcam, Boston, MA, USA), Mfn2 (1:1000; no. M6319; Sigma Aldrich), and β-actin-HRP (1:5000; no. sc-47778; Santa Cruz Inc.).

Techniques: Western Blot, Expressing, Cell Culture, Incubation

Journal: Annals of Dermatology

Article Title: Inhibitory Effect of Mitoxantrone on Collagen Synthesis in Dermal Fibroblasts

doi: 10.5021/ad.2022.34.3.206

Figure Lengend Snippet: Effect of mitoxantrone on transforming growth factor-β (TGF-β)/SMAD signaling. (A) Fibroblasts were treated with mitoxantrone for 1 hour then stimulated with TGF-β for 1 hour. Phosphorylations of SMAD2 and SMAD3 were determined by Western blot. Mitoxantrone slightly inhibited TGF-β-induced phosphorylation of SMAD3. (B) The protein levels of collagen type I α1 chain (COL1A1) and collagen type I α2 chain (COL1A2) were determined by Western blot. Mitoxantrone weakly but significantly inhibited TGF-β-induced collagen synthesis. Actin beta (ACTB) was used as a loading control.

Article Snippet: The following primary antibodies were used in this study: COL1A1 (sc-8783), COL1A2 (sc-8786), HNRNPK (sc-28380), actin beta (ACTB) (sc-47778) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA); LARP6 (H00055323-B01P; Abnova, Taipei, Taiwan); phosphorylated-SMAD family member 2 (p-SMAD2) (3108S), phosphorylated-SMAD family member 3 (p-SMAD2) (9520S) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 1. Lipopolysaccharide downregulates GILZ expression in RMECs in a dose- and time-dependent manner. (A, B) Dose-dependent effect of LPS on GILZ expression. Retinal microvascular endothelial cells were treated with 0, 10, 100, or 1000 ng/mL LPS for 24 hours and GILZ protein expression was determined by Western blotting. (C, D) Time-dependent effects of LPS on GILZ expression. Retinal microvascular endothelial cells were treated with 1000 ng/mL LPS for 0, 2, or 6 hours and GILZ protein expression was determined by Western blot analysis; b-actin was used as the loading control. Quantitative analysis of GILZ, as determined by densitometric analysis, expressed as a ratio of b-actin. Data represent means 6 SE, ANOVA with a Bonferroni post hoc test was used. n ¼ 3 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: Expressing, Western Blot, Control

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 2. Glucocorticoid-induced leucine zipper overexpression inhibits LPS-induced NF-jB p65 nuclear translocation in RMECs. (A, B) Western blotting analysis of GILZ in RMECs transfected with overexpressing recombinant lentivirus (OE-GILZ-rLV) or blank-rLV for 72 hours. (C, D) Western blotting analysis of cytosolic p65 expression in blank-rLV or OE-GILZ-rLV transfected RMECs stimulated with LPS (1 hour at 1000 ng/mL). (E, F) Western blotting analysis of nuclear p65 expression in blank-rLV or OE-GILZ-rLV transfected RMECs stimulated with LPS (1 hour at 1000 ng/mL). (G) The localization of NF-jB p65 in blank-rLV or OE-GILZ-rLV transfected RMECs stimulated with LPS (1 hour at 1000 ng/mL) was assessed by immunofluorescence. Red indicates p65-stained and blue indicates DAPI-stained nuclei. Scale bar: 10 lm. b-actin was used as the loading control of cytosolic p65; Lamin B was used as the loading control of nuclear p65. Quantitative analysis, as determined by densitometric analysis, expressed as a ratio of loading control. Data represent means 6 SE, ANOVA with a Bonferroni post hoc test was used for multiple groups. Mann-Whitney U test was used when two groups were compared. n ¼ 3 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: Over Expression, Translocation Assay, Western Blot, Transfection, Recombinant, Expressing, Staining, Control, MANN-WHITNEY

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 3. Glucocorticoid-induced leucine zipper overexpression inhibits LPS-induced NF-jB p65 phosphorylation but does not affect LPS-induced IjBa degradation in RMECs. (A, B) Western blotting analysis of IjBa expression in RMECs transfected with blank-rLV or OE-GILZ-rLV at 0, 15, 30, 45, and 60 minutes after LPS stimulation (1000 ng/mL). (C, D) Western blotting analysis of phosphorylated p65 (Ser536) in blank-rLV– or OE-GILZ-rLV– transfected RMECs stimulated with LPS (1 hour at 1000 ng/mL). b-actin was used as the loading control. Quantitative analysis, as determined by densitometric analysis, expressed as a ratio of b-actin. Data represent means 6 SE. The Student’s two-tailed t-test was used to compare the difference of IjBa between OE-GILZ-rLV with blank-rLV groups at each time point. Analysis of variance with a Bonferroni post hoc test was used for multiple comparison of phosphorylated p65 (Ser536). n ¼ 3 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: Over Expression, Phospho-proteomics, Western Blot, Expressing, Transfection, Control, Two Tailed Test, Comparison

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 6. Glucocorticoid-induced leucine zipper silencing enhances LPS-induced ICAM-1 and MCP-1 expression in RMECs. Western blotting analysis was performed to determine the protein expressions levels of GILZ (A, B), ICAM-1 (C, D), and MCP-1 (E, F) in RMECs transfected with blank-rLV or shRNA-GILZ-rLV and stimulated with LPS (24 hours at 1000 ng/mL). b-actin was used as the loading control. Quantitative analysis, as determined by densitometric analysis, expressed as a ratio of b-actin. Data represent means 6 SE, ANOVA with a Bonferroni post hoc test was used. n ¼ 3 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: Expressing, Western Blot, Transfection, shRNA, Control

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 5. Glucocorticoid-induced leucine zipper overexpression inhibits LPS-induced ICAM-1 and MCP-1 expression in RMECs. Western blotting analysis was performed to determine the protein expression levels of GILZ (A, B), ICAM-1 (C, D), and MCP-1 (E, F) in RMECs transfected with blank recombinant lentivirus (blank-rLV) or OE-GILZ-rLV and stimulated with LPS (24 hours at 1000 ng/mL). b-actin was used as the loading control. Quantitative analysis, as determined by densitometric analysis, expressed as a ratio of b-actin. Data represent means 6 SE, ANOVA with a Bonferroni post hoc test was used. n ¼ 3 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Recombinant, Control

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 7. Exogenous GILZ regulates ICAM-1 and MCP-1 expression in RMECs. The concentrations of ICAM-1 (A) and MCP-1 (B) in the culture supernatants were measured by ELISAs of RMECs transfected with blank-rLV, shRNA-GILZ-rLV, or OE-GILZ-rLV and stimulated with LPS (24 hours at 1000 ng/mL). An equal amount of PBS was added to the culture medium as a control. b-actin was used as the loading control. Quantitative analysis, as determined by densitometric analysis, expressed as a ratio of b-actin. Data represent means 6 SE, ANOVA with a Bonferroni post hoc test was used. n ¼ 20 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: Expressing, Transfection, shRNA, Control

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 8. Isoliensinine-induced dephosphorylation of NF-jB p65 attenuates LPS-induced expression of ICAM-1 and MCP-1 in RMECs. (A, B) Western blotting analysis of phosphorylated-p65 (Ser536) in RMECs treated with PBS, LPS, or LPS plus isoliensinine (ISO). (C, D) Western blotting of phosphorylated-p65 (Ser536) in RMECs transfected with blank-rLV or OE-GILZ-rLV and treated for 24 hours with LPS or LPS plus isoliensinine. Western blotting of ICAM-1 (E, F) and MCP-1 (G, H) expression in RMECs transfected with blank-rLV or sh-GILZ-rLV and treated for 24 hours with LPS or LPS plus isoliensinine. b-actin was used as the loading control. Quantitative analysis, as determined by densitometric analysis, expressed as a ratio of b-actin. Data represent means 6 SE, ANOVA with a Bonferroni post hoc test was used. n ¼ 3 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: De-Phosphorylation Assay, Expressing, Western Blot, Transfection, Control

Journal: Investigative ophthalmology & visual science

Article Title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.

doi: 10.1167/iovs.16-20933

Figure Lengend Snippet: FIGURE 9. Exogenous GILZ regulates retinal ICAM-1 and MCP-1 expression in vivo. Western blotting was performed to determine the retinal expression levels of GILZ (A, B), ICAM-1 (C, D), and MCP-1 (E, F). Viruses (OE-GILZ-rLV or blank-rLV) were intravitreally injected on day 0 and LPS (125 ng/lL, 2 lL) was intravitreally injected on day 3 and retinas were harvested on day 4. b-actin was used as the loading control. Quantitative analysis, as determined by densitometric analysis, expressed as a ratio of b-actin. Data represent means 6 SE, ANOVA with a Bonferroni post hoc test was used. n ¼ 3 for each group. *P < 0.05, **P < 0.01.

Article Snippet: The membranes were blocked in 5% nonfat milk at room temperature for 1 hour and were then incubated with the following antibodies: rabbit anti-GILZ polyclonal antibody (1:500; Proteintech, Chicago, IL, USA), anti-MCP1 antibody (ab25124; Proteintech), anti-ICAM1 antibody 1A29 (ab171123; Proteintech), rabbit anti-p65 polyclonal antibody (1:500; Proteintech), phospho-NF-jB p65 (Ser536) rabbit monoclonal antibody (1:1000; Cell Signaling Technology), inhibitory jBa (IjBa) antibody(1:500) (Proteintech), or rabbit anti-b-actin antibody (1:1,000; Abcam, Cambridge, MA, USA) overnight.

Techniques: Expressing, In Vivo, Western Blot, Injection, Control