Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Extracellular matrix (ECM) contraction assay. Human colonic CCD-18Co fibroblasts were embedded in 100 μL/well of Matrigel (Corning #356234) and covered by 1 mL/well serum-free minimal essential medium (MEM). TGF-β1 (10 ng/mL) and elafin (1 μg/mL) were added to the medium on day 0. The diameter of the Matrigel was measured on day 9. TGF-β1 treatment significantly reduced the Matrigel diameter, indicating increased ECM stiffness. Shrinkage was unaffected by elafin. Results were pooled from 3 independent experiments. Ordinary one-way ANOVA with Tukey test. ( B ) Human colonic CCD-18Co fibroblasts were treated with elafin for 48 hours, followed by addition of MTS assay reagent (G5421; Promega, Madison, WI). Absorbance was determined at 490 nm. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( C ) Serum-starved primary human colonic epithelial cells were pretreated with TGF-β1 (10 ng/mL) or CDSE (100 μg/mL) for 2 hours and then incubated with elafin for 48 hours. N-cadherin, ZEB1, and COL1A2 mRNA expression was determined by real-time reverse transcription polymerase chain reaction. TGF-β1, but not CDSE, induced N-cadherin, ZEB1, and COL1A2 mRNA expression, unaffected by elafin. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Contraction Assay, MTS Assay, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A and B ) Serum-starved CD-HIF were pretreated with 0.8% DMSO, 10 μmol/L PAR2 inhibitor GB88 (HY-120261; MCE), 10 μmol/L PAR1 agonist TRAP-6 (HY-P0078; MCE), or 10 μmol/L PAR2 agonist SLIGKV-NH 2 (HY-P0283; MCE). An hour later, the fibroblasts were exposed to 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. Results were pooled from 4 experiments. Ordinary one-way ANOVA with Tukey test. ( C ) The most differentially expressed genes found in whole-transcriptome RNA sequencing in the colonic tissues of 2 stricturing and 2 non-stricturing CD patients. ( D ) STRING database analysis shows protein interaction association between the most differentially expressed genes in stricturing versus non-stricturing CD patients. ( E, upper panel ) Colonic ZEB1 mRNA expression in 40 non-IBD, 52 UC, 28 non-stricturing CD, and 15 stricturing CD patients. Stricturing CD patients have significantly higher colonic ZEB1 mRNA expression than non-stricturing CD patients. Ordinary one-way ANOVA with Tukey test. ( E, lower panel ) Positive correlation between colonic ZEB1 mRNA and collagen (ProCOL1A1) protein expression in 43 CD patients. ( F ) Serum-starved CD-HIF were transfected with either control (sc-37007; Santa Cruz Biotechnology, Dallas, TX) or ZEB1 (sc-38643; Santa Cruz Biotechnology) siRNA via lipofectamine 3000 overnight, followed by 100 μg/mL CDSE for 24 hours. ZEB1 and ProCOL1A1 proteins were measured by ELISA. Results were pooled from 4 experiments. Ordinary one-way ANOVA with Tukey test for left panel. Student t test was used to compare ZEB1 expression between control siRNA and ZEB1 siRNA groups on right panel.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Incubation, RNA Sequencing Assay, Expressing, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Serum-starved CD-HIF were transfected with either control or ZEB1-overexpressing construct via lipofectamine 3000 overnight. Fibroblasts were then pretreated with 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( B ) Efficiency of ZEB1-overexpressing construct transfection was determined by ELISA. Results were pooled from 4 experiments. Student t test was used to compare ZEB1 expression between control construct and ZEB1-overexpressing construct groups. ( C ) Serum-starved CD-HIF were pretreated with DMSO, 0.4 μg/mL cathepsin S, 10 μmol/L PAR1 agonist TRAP-6 (HY-P0078; MCE), or 10 μmol/L PAR2 agonist (HY-P0283; MCE) for 60 minutes, followed by addition of 100 μg/mL CDSE. Two hours later, some groups were treated with elafin (1 μg/mL) and incubated for 24 hours. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( D ) Fresh human colonic tissues from 4 colon cancer patients were incubated in serum-free RPMI1640 media with or without 100 μg/mL CDSE. Two hours later, elafin was added and further incubated for 24 hours. Results were pooled from 4 independent experiments. Ordinary one-way ANOVA with Tukey test. ( E ) Fresh human ileal tissues from 3 stricturing CD patients were incubated in serum-free RPMI1640 media with or without 100 μg/mL CDSE. Two hours later, elafin (1 μg/mL) was added and incubated for 24 hours. COL1A1, COL1A2, and ZEB1 mRNA expression were determined by real-time reverse transcription polymerase chain reaction. Ordinary one-way ANOVA with Tukey test. ( F ) Macroscopic and microscopic morphology of the fresh stricturing ileal tissue. Intense collagen deposition is found in the mucosal layer.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

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

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Experimental plan. Control lentivirus, elafin-overexpressing lentiviruses, Ctss-overexpressing lentivirus, Ctss-siRNA lentivirus, Zeb1-shRNA lentivirus, and Zeb1-overexpressing lentivirus were injected into SAMP1/YitFc mice intraperitoneally once at 40 weeks of age. In addition, PAR2 agonist GB110 or PAR2 inhibitor GB88 was given via oral gavage from 40 to 42 weeks of age. Non-fibrotic 10-week-old SAMP1/YitFc mice and parental control 42-week-old AKR strain mice were used for comparison. Ileal tissues were collected for analysis 2 weeks after lentiviral injection. ( B ) Body weight. Six mice per group. Mean ± standard deviation. ( C ) H&E staining ( upper panels ) and Masson Trichrome (MT) staining ( lower panels ) of ileal tissues from 10 to 42 weeks of age. Blue color in MT staining ( arrows ) indicated collagen deposition in lamina propria. ( D ) Ileal histology scores. ( E ) Ileal fibrosis scores. ( F ) Ileal overall disease activities. Six mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: shRNA, Injection, Standard Deviation, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: Ileal Overall Disease Activity and Gene Expression Profile in SAMP1/YitFc Model

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Activity Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Experimental plan. Eight-week-old male and female 129Sv/J mice were administered 20 mg streptomycin via oral gavage. Twenty-four hours later, mice were orally infected with Salmonella typhimurium SL1344 strain (1 × 10 8 colony-forming units) to induce cecal fibrosis. In addition, some mice received single intraperitoneal injection (10 infectious units/mouse) of control lentivirus or elafin-overexpressing lentiviruses on day 14. Cecal tissues were collected for analysis on day 21. ( B ) No significant change in body weight was noticed throughout the disease course in the infected mice. Six mice per group. Mean ± standard deviation. ( C ) H&E staining ( upper panels ) and Masson Trichrome (MT) staining ( lower panels ) of cecal tissues on day 21. Blue color in MT staining ( arrows ) indicated collagen deposition in cecal lamina propria of Salmonella -infected mice. ( D ) Cecal histology scores. ( E ) Cecal fibrosis scores. ( F ) Cecal overall disease activities. Lentiviral elafin expression reversed cecal fibrosis in Salmonella -infected mice. Six mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Infection, Injection, Standard Deviation, Staining, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: Cecal Overall Disease Activity and Gene Expression Profile in Salmonella Model

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Activity Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: Colonic Overall Disease Activity and Gene Expression Profile in TNBS Model

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Activity Assay, Expressing, Construct

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) H&E staining ( upper panels ) and MT staining ( lower panels ) of ileal tissues from SAMP1/YitFc mice at 42 weeks of age. Blue color in MT staining ( arrows ) indicated collagen deposition. ( B ) Ileal histology scores. ( C ) Ileal fibrosis scores. ( D ) Ileal overall disease activities. Prominent ileal fibrosis was found in elafin-overexpressing groups with lentiviral Ctss and Zeb1 overexpression and oral PAR2 agonist GB110 treatment. Ileal fibrosis was ameliorated with lentiviral Ctss and Zeb1 shRNA inhibition and oral PAR2 inhibitor GB88 treatment. Six AKR or SAMP1/YitFc mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Staining, Over Expression, shRNA, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) H&E staining ( upper panels ) and Masson Trichrome (MT) staining ( lower panels ) of cecal tissues on day 21. Blue color in MT staining ( arrows ) indicated collagen deposition in cecal lamina propria of Salmonella -infected mice. Prominent cecal fibrosis was found in elafin-overexpressing groups with lentiviral Ctss and Zeb1 overexpression and oral PAR2 agonist GB110 treatment. Cecal fibrosis was ameliorated with lentiviral Ctss and Zeb1 shRNA inhibition and oral PAR2 inhibitor GB88 treatment. ( B ) Cecal histology scores. ( C ) Cecal fibrosis scores. ( D ) Cecal overall disease activities. Six uninfected or Salmonella -infected mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Staining, Infection, Over Expression, shRNA, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) TNBS-treated mice were injected with Ctss-overexpressing lentivirus, Ctss-siRNA lentiviruses, Zeb1-overexpressing lentivirus, or Zeb1-shRNA lentivirus on day 9 after last TNBS injection. Some mice were injected with 5 mg/kg PAR2 agonist SLIGKV-NH 2 intracolonically 9, 11, and 13 days after last TNBS injection. GB88 (10 mg/kg/day) was administered via oral gavage. Control or elafin-overexpressing construct was injected intracolonically. H&E staining ( upper panels ) and Masson Trichrome staining ( lower panels ) of colonic tissues are shown. Blue color in MT staining ( arrows ) indicated collagen deposition. ( B ) Colonic histology scores. ( C ) Colonic fibrosis scores. ( D ) Colonic overall disease activities. Prominent colonic fibrosis was found in elafin-overexpressing groups with lentiviral Ctss and Zeb1 overexpression and oral PAR2 agonist GB110 treatment. Colonic fibrosis was ameliorated with lentiviral Ctss and Zeb1 shRNA inhibition and oral PAR2 inhibitor GB88 treatment. Six mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Injection, shRNA, Construct, Staining, Over Expression, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: Comparison of Overall Disease Activities and Gene Expression in Mice With Lentiviral and Pharmacologic Manipulations

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: Elafin-overexpressing lentiviruses, Ctss-overexpressing lentivirus, and Zeb1-overexpressing lentivirus were injected into AKR mice intraperitoneally at 40 weeks of age. In addition, oral PAR2 agonist GB110 (10 mg/kg/day) was administered via oral gavage from 40 to 42 weeks of age. H&E staining ( upper panels ) and Masson Trichrome (MT) staining ( lower panels ) of ileal tissues did not find histologic injury or fibrosis. Six mice per group.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Injection, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Experimental plan. The formulation was suspended in mildly acidified (pH 5) water containing 0.5% hydroxypropyl methylcellulose (HPMC) and administered to TNBS-treated mice via oral gavage. The Eudragit polymer releases its drug in the mid-distal ileum and colon under an alkaline environment at ∼pH 8. ( B ) Elafin-Eudragit-HPMC (10 mg/kg) was administered to normal mice via oral gavage. Colonic tissue elafin levels were determined by ELISA (DY1747; R&D Systems). Mean ± standard deviation. ( C ) H&E staining ( upper panels ) and MT staining ( lower panels ) of colonic tissues. Blue color in MT staining ( arrows ) indicated collagen deposition. ( D ) Histology scores, fibrosis scores, overall disease activities, colonic Zeb1 mRNA expression, and changes in body weight. Six mice per group. Ordinary one-way ANOVA with Tukey tests. ( E ) Comparison of intestinal tissue elafin levels and efficiencies in regulating target genes and disease activities. Body weight changes in elafin treatment groups compared with their respective positive control groups. Mean ± standard deviation.

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Staining, Expressing, Positive Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: Catalog and Batch Numbers of Reagents

Article Snippet: TNBS-treated mice were injected with Ctss-overexpressing lentivirus (#171210640196) or Ctss-siRNA lentiviruses (#171210940296; Applied Biological Materials, Inc), Zeb1-overexpressing lentivirus (MR223095L2V), or Zeb1-shRNA lentivirus (TL513177V; OriGene) on the ninth day after the last TNBS injection.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Experimental plan. Control lentivirus, elafin-overexpressing lentiviruses, Ctss-overexpressing lentivirus, Ctss-siRNA lentivirus, Zeb1-shRNA lentivirus, and Zeb1-overexpressing lentivirus were injected into SAMP1/YitFc mice intraperitoneally once at 40 weeks of age. In addition, PAR2 agonist GB110 or PAR2 inhibitor GB88 was given via oral gavage from 40 to 42 weeks of age. Non-fibrotic 10-week-old SAMP1/YitFc mice and parental control 42-week-old AKR strain mice were used for comparison. Ileal tissues were collected for analysis 2 weeks after lentiviral injection. ( B ) Body weight. Six mice per group. Mean ± standard deviation. ( C ) H&E staining ( upper panels ) and Masson Trichrome (MT) staining ( lower panels ) of ileal tissues from 10 to 42 weeks of age. Blue color in MT staining ( arrows ) indicated collagen deposition in lamina propria. ( D ) Ileal histology scores. ( E ) Ileal fibrosis scores. ( F ) Ileal overall disease activities. Six mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: Control lentivirus (PS100064V; OriGene, Rockville, MD), elafin-overexpressing lentiviruses (RC203136L1V; OriGene), Ctss-overexpressing lentivirus (#171210640196; Applied Biological Materials, Richmond, BC, Canada), Ctss-siRNA lentivirus (#171210940296; Applied Biological Materials), Zeb1-shRNA lentivirus (TL513177V; OriGene), and Zeb1-overexpressing lentivirus (MR223095L2V; OriGene) were injected to AKR or SAMP1/YitFc mice intraperitoneally once at 40 weeks of age.

Techniques: shRNA, Injection, Standard Deviation, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) Experimental plan. Eight-week-old male and female 129Sv/J mice were administered 20 mg streptomycin via oral gavage. Twenty-four hours later, mice were orally infected with Salmonella typhimurium SL1344 strain (1 × 10 8 colony-forming units) to induce cecal fibrosis. In addition, some mice received single intraperitoneal injection (10 infectious units/mouse) of control lentivirus or elafin-overexpressing lentiviruses on day 14. Cecal tissues were collected for analysis on day 21. ( B ) No significant change in body weight was noticed throughout the disease course in the infected mice. Six mice per group. Mean ± standard deviation. ( C ) H&E staining ( upper panels ) and Masson Trichrome (MT) staining ( lower panels ) of cecal tissues on day 21. Blue color in MT staining ( arrows ) indicated collagen deposition in cecal lamina propria of Salmonella -infected mice. ( D ) Cecal histology scores. ( E ) Cecal fibrosis scores. ( F ) Cecal overall disease activities. Lentiviral elafin expression reversed cecal fibrosis in Salmonella -infected mice. Six mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: Control lentivirus (PS100064V; OriGene, Rockville, MD), elafin-overexpressing lentiviruses (RC203136L1V; OriGene), Ctss-overexpressing lentivirus (#171210640196; Applied Biological Materials, Richmond, BC, Canada), Ctss-siRNA lentivirus (#171210940296; Applied Biological Materials), Zeb1-shRNA lentivirus (TL513177V; OriGene), and Zeb1-overexpressing lentivirus (MR223095L2V; OriGene) were injected to AKR or SAMP1/YitFc mice intraperitoneally once at 40 weeks of age.

Techniques: Infection, Injection, Standard Deviation, Staining, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: ( A ) TNBS-treated mice were injected with Ctss-overexpressing lentivirus, Ctss-siRNA lentiviruses, Zeb1-overexpressing lentivirus, or Zeb1-shRNA lentivirus on day 9 after last TNBS injection. Some mice were injected with 5 mg/kg PAR2 agonist SLIGKV-NH 2 intracolonically 9, 11, and 13 days after last TNBS injection. GB88 (10 mg/kg/day) was administered via oral gavage. Control or elafin-overexpressing construct was injected intracolonically. H&E staining ( upper panels ) and Masson Trichrome staining ( lower panels ) of colonic tissues are shown. Blue color in MT staining ( arrows ) indicated collagen deposition. ( B ) Colonic histology scores. ( C ) Colonic fibrosis scores. ( D ) Colonic overall disease activities. Prominent colonic fibrosis was found in elafin-overexpressing groups with lentiviral Ctss and Zeb1 overexpression and oral PAR2 agonist GB110 treatment. Colonic fibrosis was ameliorated with lentiviral Ctss and Zeb1 shRNA inhibition and oral PAR2 inhibitor GB88 treatment. Six mice per group. Ordinary one-way ANOVA with Tukey tests.

Article Snippet: Control lentivirus (PS100064V; OriGene, Rockville, MD), elafin-overexpressing lentiviruses (RC203136L1V; OriGene), Ctss-overexpressing lentivirus (#171210640196; Applied Biological Materials, Richmond, BC, Canada), Ctss-siRNA lentivirus (#171210940296; Applied Biological Materials), Zeb1-shRNA lentivirus (TL513177V; OriGene), and Zeb1-overexpressing lentivirus (MR223095L2V; OriGene) were injected to AKR or SAMP1/YitFc mice intraperitoneally once at 40 weeks of age.

Techniques: Injection, shRNA, Construct, Staining, Over Expression, Inhibition

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2

doi: 10.1016/j.jcmgh.2022.06.011

Figure Lengend Snippet: Elafin-overexpressing lentiviruses, Ctss-overexpressing lentivirus, and Zeb1-overexpressing lentivirus were injected into AKR mice intraperitoneally at 40 weeks of age. In addition, oral PAR2 agonist GB110 (10 mg/kg/day) was administered via oral gavage from 40 to 42 weeks of age. H&E staining ( upper panels ) and Masson Trichrome (MT) staining ( lower panels ) of ileal tissues did not find histologic injury or fibrosis. Six mice per group.

Article Snippet: Control lentivirus (PS100064V; OriGene, Rockville, MD), elafin-overexpressing lentiviruses (RC203136L1V; OriGene), Ctss-overexpressing lentivirus (#171210640196; Applied Biological Materials, Richmond, BC, Canada), Ctss-siRNA lentivirus (#171210940296; Applied Biological Materials), Zeb1-shRNA lentivirus (TL513177V; OriGene), and Zeb1-overexpressing lentivirus (MR223095L2V; OriGene) were injected to AKR or SAMP1/YitFc mice intraperitoneally once at 40 weeks of age.

Techniques: Injection, Staining

Journal: Nature communications

Article Title: Metastasis is regulated via microRNA-200/ZEB1 axis control of tumor cell PD-L1 expression and intratumoral immunosuppression

doi: 10.1038/ncomms6241

Figure Lengend Snippet: ( a ) Representative FACS histogram of PD-L1 expression on murine (344SQ, 531LN2, and 393P) and human (H157, H1155, and HCC827) lung cancer cells by overexpression of miR-200 and Zeb1. The analysis was independently repeated three times. ( b ) The representative FACS histogram of PD-L1 expression on murine lung cancer cells co-cultured with 129/Sv murine splenocytes with or without IFN-γ neutralizing antibody. Long dash is isotype control staining; blue line is anti-PD-L1 staining without splenocytes; green line is anti-PD-L1 staining with splenocytes blocked by the neutralizing antibody to IFN-γ (20 μg ml −1 , 48 hrs); red line is anti-PD-L1 staining with splenocytes (48 hrs). The assay was independently performed three times. ( c ) The representative FACS histogram of PD-L1 (MFI, mean fluorescence intensity) expression in primary subcutaneous tumors grown in syngeneic 129/Sv mice (n = 3) injected with the indicated cell lines shown in the upper panel. The representative PD-L1 IHC staining of each tumor type shown in the bottom panel. Samples were obtained 2 weeks post-cell injection. Scale bar, 100 μm. ( d ) 3’-UTR luciferase reporter assay for wild-type PD-L1, single (mutant A or B) and double mutants (A and B) versus a Zeb1 3’-UTR control in murine 344SQ cells. The reporters were transiently co-transfected with synthetic miR-200 precursors (200a, 200b, or 200c) or control oligomers (con) into 344SQ cells. Values (p < 0.05) were normalized based on renilla luciferase and expressed as the mean values (±S.D.) of triplicate wells relative to that of controls co-transfected with empty reporter and empty expression vector or scrambled precursors, which were set at 1.0. The target sequences are shown in the top panel, with the introduced mutations highlighted in red.

Article Snippet: Mouse ZEB1 was subcloned into pcDNA3.1/His C (Invitrogen) to generate the ZEB1 overexpression vector. shRNA to mouse ZEB1 and PD-L1 and scrambled controls in the pGFP-V-RS vector were obtained from OriGene.

Techniques: Expressing, Over Expression, Cell Culture, Staining, Fluorescence, Injection, Immunohistochemistry, Luciferase, Reporter Assay, Mutagenesis, Transfection, Plasmid Preparation

Journal: Nature communications

Article Title: Metastasis is regulated via microRNA-200/ZEB1 axis control of tumor cell PD-L1 expression and intratumoral immunosuppression

doi: 10.1038/ncomms6241

Figure Lengend Snippet: ( a , b ) FACS analysis of ( a ) CD8 + TIL frequency; ( b ) PD1 and TIM3 marker expression on CD8 + T cells from 393P_vector and 393P_ZEB1 (n = 5), as well as 344SQ_vector and 344SQ_miR-200 (n = 10) primary tumors. Analysis was done 2 weeks post-cancer cell injection. ( c , d ) ( c ) Intratumoral Ki67 + CD8 + T cells; ( d ) granzyme B (GzB) + CD8 + T cells in 344SQ_vector or 344SQ_miR-200 primary tumors 6 weeks post-subcutaneous injection of cancer cells into 129/Sv mice. Representative Ki67 or GzB staining in an individual tumor sample is shown on the left, and mean Ki67 + or GzB + populations of gated CD8 + T cells in total T cells are shown on the right (n = 5). ( e ) CD8 + T cell depletion results in tumor growth and metastasis in mice (n = 5) that received subcutaneous tumor cell injections. No treatment (344SQ_vector (Vector)), IgG (344SQ_miR-200 + IgG control), or Ab (344SQ_miR-200 + anti-CD8 Ab). The analysis was done 6 weeks post-injection. ( f ) Relative abundance of CD8 + T cells in the tumor (left) or lung (right) from 129/Sv mice (n =5) with syngeneic control 344SQ tumors (Vector), 344SQ_miR-200 tumors with control IgG treatment (IgG) or anti-CD8 antibody treatment (Ab). ( g ) Lung metastases of 344SQ_vector (Vector) and 344SQ_miR-200 (miR-200) tumors in wild-type (WT) or 129/Sv Rag2 −/− ( Rag2 −/− ) mice (n = 5). The analysis was done 6 weeks post-tumor cell subcutaneous injection. All the analyses were independently repeated twice. Data are shown as mean ± s.e.m. t -test was used to analyze, with P values shown in the graphs.

Article Snippet: Mouse ZEB1 was subcloned into pcDNA3.1/His C (Invitrogen) to generate the ZEB1 overexpression vector. shRNA to mouse ZEB1 and PD-L1 and scrambled controls in the pGFP-V-RS vector were obtained from OriGene.

Techniques: Marker, Expressing, Plasmid Preparation, Injection, Staining

Journal: Nature communications

Article Title: Metastasis is regulated via microRNA-200/ZEB1 axis control of tumor cell PD-L1 expression and intratumoral immunosuppression

doi: 10.1038/ncomms6241

Figure Lengend Snippet: ( a ) The knockdown (KD) efficiency of PD-L1 in LLC-JSP murine lung cancer cells measured by FACS. Representative histograms (left), and statistical analysis (right). The measurement was independently repeated at least three times. ( b ) Representative FACS histogram of PD-L1 expression on myeloid cells (CD11b + ) in PD-L1 KO or WT mice (n = 3). ( c ) Tumor growth in PD-L1 KO or WT mice (n = 6) of subcutaneously injected LLC-JSP cells (10, 000 cells with 100 μl of PBS per mouse) with differing PD-L1 knockdown (Vector, PD-L1 KD vector control; PD-L1 int , PD-L1 intermediate KD; PD-L1 hi , PD-L1 high level KD). The data is shown from two independent experiments. Data are shown as mean ± s.e.m. ( d ) FACS analysis of CD8 + TIL frequency and T cell exhaustion marker expression levels on CD8 + T cells in subcutaneous primary tumors of LLC-JSP vector control (Vector ctrl) and LLC-JSP intermediate PD-L1 knockdown (Intermediate KD) in PD-L1 WT (WT) and PD-L1 KO (KO) mice (n = 5, from two independent experiments). Analysis was done 3 weeks post-tumor cell injection (20, 000 cells with 100 μl of PBS per mouse). t -test was used to analyze the data. p < 0.0001. ( e ) PD-L1 expression levels on LLC-JSP-shPD-L1 cells (the high knockdown efficiency cells) after reconstitution of PD-L1. The measurement was independently repeated at least three times. Control, LLC-JSP-shPD-L1 + vector control; PD-L1 medium , LLC-JSP-shPD-L1 + intermediate overexpression of PD-L1; PD-L1 high , LLC-JSP-shPD-L1 + high overexpression of PD-L1. ( f ) Primary tumor masses and lung metastases in PD-L1 KO or WT mice (n = 5) injected subcutaneously with the different reconstituted cell lines. Analysis was done 4 weeks post-subcutaneous cancer cell injection (10, 000 cells with 100 μl of PBS per mouse). The analyses were independently repeated twice. Data are shown as mean ± s.e.m. t -test was used to analyze the data. ns, p > 0.05. ( g ) The working model of the mutual regulation of EMT and immune suppression by miR-200/ZEB1 axis and their complementary role in metastasis.

Article Snippet: Mouse ZEB1 was subcloned into pcDNA3.1/His C (Invitrogen) to generate the ZEB1 overexpression vector. shRNA to mouse ZEB1 and PD-L1 and scrambled controls in the pGFP-V-RS vector were obtained from OriGene.

Techniques: Expressing, Injection, Plasmid Preparation, Marker, Over Expression

Journal: Nature Communications

Article Title: Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation

doi: 10.1038/s41467-023-43896-7

Figure Lengend Snippet: a Feeding protocol schematic for Zeb1 (+/+)/ ApoE KO and Zeb1 (+/−)/ ApoE KO male mice. From birth until 8 weeks of age, mice were provided ad libitum access to the Chow diet, followed by 12 weeks on the Western diet. b Total bodyweight of Zeb1 (+/+)/ ApoE KO and Zeb1 (+/−)/ ApoE KO mice at the end of the protocol before euthanasia ( n = 5). c Representative captures and quantification of the atherosclerotic lesion area in aortic root sections of Zeb1 (+/+)/ ApoE KO ( n = 4) and Zeb1 (+/−)/ ApoE KO (n = 5) mice at the end of the feeding protocol. d Feeding protocol schematic for Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO male mice. From birth to the age of 8-week-old, mice were provided ad libitum access to the Chow diet followed by 10 weeks on the Western diet. e Zeb1 mRNA levels in the peritoneal macrophages of Zeb1 WT / Apoe KO ( n = 7) and Zeb1 ∆M / Apoe KO ( n = 9) mice. f Representative en face ORO staining images and atherosclerosis lesion area quantification in Zeb1 WT / Apoe KO ( n = 11) and Zeb1 ∆M / Apoe KO ( n = 10) mice at the end of the Western diet feeding protocol. g As in f , representative images and quantification of the atherosclerosis lesion area of aortic root sections stained with H&E from mice of both genotypes. The dashed line delineates the plaque and “N” indicates a representative area of necrosis. Scale bar: 200 μm. ( n = 7,9). h As in g , but sections were stained with ORO to assess lipid accumulation. Scale bar: 200 μm. ( n = 9,9). i As in g , but sections were stained with BODIPY® 493/503 (hereinafter referred to as BODIPY). Scale bar: 50 μm. ( n = 7, 6). j As in g , but sections were stained for LAMP2/MAC3 (clone M3/84, dilution 1/50). Scale bar: 50 μm ( n = 8,7). k As in g , but tissue sections were stained for Sirius Red. Scale bar: 100 μm. ( n = 6, 7). l Quantification of plaque necrosis from the staining for Sirius Red. ( n = 6,7). m As in g , but sections were stained with TUNEL and LAMP2/MAC3 (clone M3/84, dilution 1/50) along with DAPI for nuclear staining. Quantification of “macrophage-associated” apoptotic cells to “free AC”. Scale bar: 25 μm. ( n = 5,6). All Graphs represent mean values ± SEM with two-tailed unpaired Mann–Whitney test. p ≤ 0.001 (***), p ≤ 0.01 (**) or p ≤ 0.05 (*) levels, or non-significant (ns) for values of p > 0.05, and with specified numerical p values for 0.05 < p < 0.075. The raw data, along with p values from the statistical analyses, are included in the Source Data file.

Article Snippet: The development of the conditional Zeb1 flox allele mouse ( Zeb1 fl/fl , which is herein designated as Zeb1 WT ) was carried out at the Transgenesis Unit, which is jointly affiliated with the National Biotechnology Center (CNB) and the Severo Ochoa’s Molecular Biology Center-Autonomous University of Madrid (CBMSO-UAM) (Madrid, Spain), both centers belonging to the Spanish National Research Council (CSIC).

Techniques: Western Blot, Staining, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation

doi: 10.1038/s41467-023-43896-7

Figure Lengend Snippet: a Macrophage subpopulations were analyzed using published scRNAseq datasets of mouse atherosclerosis (GSE116240 and GSE149070) , , . b The share of macrophages out of CD45 + cells within the atherosclerotic plaque of Zeb1 WT / Apoe KO ( n = 4) and Zeb1 ∆M / Apoe KO ( n = 5) mice was determined by FACS. c As in b , but the share of CD86 high macrophages was calculated out of the total number of macrophages (CD45 + , CD11b + , F4/80 + ). ( n = 3,4). d As in b , but the share of CD86 high macrophages was calculated out of total CD45 + cells. ( n = 3, 4). e Stacked bar chart for ( c ) and ( d ). f – h As in c – e , but for CD9 high macrophages. i Total body weight of Zeb1 WT / Apoe KO ( n = 10) and Zeb1 ∆M / Apoe KO ( n = 13) mice at the end of the feeding protocol. j The share of macrophages out of CD45 + cells infiltrating the WAT of Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO mice. ( n = 4). k and l Zeb1 and Il1b mRNA levels in the macrophages infiltrating the WAT of Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO mice ( n = 3). m Serum levels of IL1β ( n = 13), IL2 ( n = 13,12), IL12 ( n = 12,14), and CCL2 ( n = 12,13) from Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO mice at the end of the feeding protocol. n Serum levels of leptin in Zeb1 WT / Apoe KO ( n = 8) and Zeb1 ∆M / Apoe KO (n = 10) mice. o Glucose tolerance test in Zeb1 WT / Apoe KO ( n = 7) and Zeb1 ∆M / Apoe KO ( n = 6) mice at the end of the feeding protocol plus 16 h of fasting. p Liver weight of Zeb1 WT / Apoe KO ( n = 5) and Zeb1 ∆M / Apoe KO ( n = 6) mice. q Representative images of liver sections from Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO mice stained with H&E. Scale bar: 20 μm. r As in q , sections were stained for ORO. Representative images and quantification. Scale bar: 20 μm. ( n = 6). s Lysates from the livers of Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO at the end of the feeding protocol plus 12 h of fasting were blotted for mSREBP1c (clone 2A4, dilution 1/1000) and GAPDH (1E6D9, 1/20,000) as loading control. ( n = 3). t Serum levels of total cholesterol ( n = 11,13), free colesterol ( n = 8,9), HDL ( n = 7,9), and LDL ( n = 11,13) in Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO mice at the end of the feeding protocol. Graphs represent mean values ± SEM with two-tailed unpaired Student’s t test. p ≤ 0.001 (***), p ≤ 0.01 (**) or p ≤ 0.05 (*) levels, or non-significant (ns) for values of p > 0.05. The raw data, along with p values from statistical analyses are included in the Source Data file.

Article Snippet: The development of the conditional Zeb1 flox allele mouse ( Zeb1 fl/fl , which is herein designated as Zeb1 WT ) was carried out at the Transgenesis Unit, which is jointly affiliated with the National Biotechnology Center (CNB) and the Severo Ochoa’s Molecular Biology Center-Autonomous University of Madrid (CBMSO-UAM) (Madrid, Spain), both centers belonging to the Spanish National Research Council (CSIC).

Techniques: Staining, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation

doi: 10.1038/s41467-023-43896-7

Figure Lengend Snippet: a Zeb1 WT and Zeb1 ∆M peritoneal macrophages either untreated or treated with 50 μg/mL of ox-LDL for 24 h were assessed for lipid accumulation with BODIPY and Filipin. Representative images (scale bar: 20 μm) and quantification (BODIPY, n = 6,5,7,7; Filipin, n = 3). b BODIPY staining of macrophages from Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO mice at the end of the feeding protocol ( n = 8). c mRNA levels of Abca1 ( n = 4,5,5,5), Abcg1 ( n = 4,4,6,5), and ScarbI ( n = 4,3,3,3) were determined by qRT-PCR in Zeb1 WT and Zeb1 ∆M peritoneal macrophages untreated or treated for 24 h with 50 μg/mL of oxLDL. d Cholesterol efflux to lipid-free APOA1 and HDL in Zeb1 WT and Zeb1 ∆M peritoneal macrophages preloaded with 3 H-cholesterol and 50 μg/mL of oxLDL (APOA1 n = 5,6; HDL n = 4,5). e As in c , but for Prkaa1 ( n = 4,4,5,4) , Nr1h3 ( n = 4,6,6,6) , Ppargc1a ( n = 3), and Slc2a1 ( n = 4,4,3,3). f BODIPY staining in Zeb1 WT and Zeb1 ∆M peritoneal macrophages treated with 50 μg/mL of oxLDL in the presence or absence of 100 μM of A769662. Representative pictures (scale bar: 20 μm) and quantification. ( n = 3,3,4,4). g As in f , but Nr1h3 ( n = 3,3,4,3) and Ppargc1a ( n = 4,5,5,6) mRNA expression was assessed. h As in f , but Zeb1 WT and Zeb1 ∆M peritoneal macrophages treated with 50 μg/mL of oxLDL in the presence or absence of 15 μM of compound C (CC) ( n = 3,3,4,4). Graphs represent mean values ± SEM with two-tailed unpaired Student’s t test. p ≤ 0.001 (***), p ≤ 0.01 (**) or p ≤ 0.05 (*) levels, or non-significant (ns) for values of p > 0.05. The raw data, along with p values from statistical analyses are included in the Source Data file.

Article Snippet: The development of the conditional Zeb1 flox allele mouse ( Zeb1 fl/fl , which is herein designated as Zeb1 WT ) was carried out at the Transgenesis Unit, which is jointly affiliated with the National Biotechnology Center (CNB) and the Severo Ochoa’s Molecular Biology Center-Autonomous University of Madrid (CBMSO-UAM) (Madrid, Spain), both centers belonging to the Spanish National Research Council (CSIC).

Techniques: Staining, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation

doi: 10.1038/s41467-023-43896-7

Figure Lengend Snippet: a Heatmap of the top differentially expressed genes (DEG) in the RNAseq of infiltrating macrophages in the plaque of Zeb1 WT / Apoe KO ( n = 2) and Zeb1 ∆M / Apoe KO ( n = 3) mice at the end of the Western diet feeding protocol. b mRNA levels of Adss1 ( n = 3) , Chmp1b ( n = 6,6,7,7) , Asl ( n = 3), and Vps52 ( n = 3,4,4,4) were determined by qRT-PCR in Zeb1 WT and Zeb1 ∆M peritoneal macrophages untreated or treated for 24 h with 50 μg/mL of ox-LDL. c Ultrastructure of areas containing macrophages in atherosclerotic plaque in Zeb1 WT / Apoe KO and Zeb1 ∆M / Apoe KO mice at the end of the feeding protocol were examined by TEM. Two representative captures are shown and additional captures are displayed in Supplementary Fig. . Macrophages (M), cholesterol crystals (CC), late endosomes/lysosomes (E/L). Scale bar: 10 μm ( n = 3). d The uptake and transport of pHrodo™ Green-LDL fluorescence in Zeb1 WT and Zeb1 ∆M macrophages were captured over time (0–30 min) by confocal microscopy (see the corresponding Supplementary Movies and , respectively, in Supplementary Information). Representative captures. Scale bar: 10 μm. e As in d , n = 18 cells quantification of three independent experiments. f DiI-oxLDL fluorescence of Zeb1 WT and Zeb1 ∆M macrophages at 0, 30, and 60 min was assessed by confocal microscopy. See Supplementary Fig. for individual staining pictures. Representative captures. Scale bar: 10 μm. g As in f , n = 17,18 cells quantification of two independent experiments. h Representative pictures of Zeb1 WT and Zeb1 ∆M macrophages stained for NPC2 (red, clone 19888-1-AP, dilution 1/100) and LAMP1 (green, H4A3, 1/500) and assessed by confocal microscopy of two independent experiments. See Supplementary Fig. for individual staining. R epresentative captures. Scale bar: 10 μm. i Representative TEM images of Zeb1 WT and Zeb1 ∆M macrophages from a 5–6 mice pool for each genotype. In Zeb1 WT macrophages, oxLDL is internalized via receptor-mediated endocytosis, forming multivesicular bodies (MVBs, late endosomes) that fuse with lysosomes to complete degradation. In Zeb1 ∆M macrophages, cholesterol accumulates in endolysosomes and forms crystals (red arrows). Zeb1 ∆M / Apoe KO macrophages also exhibited Golgi fragmentation (dashed squares). mit mitochondria, MVB multivesicular body (late endosome), G Golgi complex, N nucleus, ER endoplasmic reticulum, Lys lysosome. Scale bar: 500 nm. Graphs represent mean values ± SEM with two-tailed unpaired Student’s t test ( b ) or two-way ANOVA test ( e , g ). p ≤ 0.001 (***), p ≤ 0.01 (**) or p ≤ 0.05 (*) levels, or non-significant (ns) for values of p > 0.05. The raw data, along with p values from statistical analyses are included in the Source Data file.

Article Snippet: The development of the conditional Zeb1 flox allele mouse ( Zeb1 fl/fl , which is herein designated as Zeb1 WT ) was carried out at the Transgenesis Unit, which is jointly affiliated with the National Biotechnology Center (CNB) and the Severo Ochoa’s Molecular Biology Center-Autonomous University of Madrid (CBMSO-UAM) (Madrid, Spain), both centers belonging to the Spanish National Research Council (CSIC).

Techniques: Western Blot, Quantitative RT-PCR, Fluorescence, Confocal Microscopy, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation

doi: 10.1038/s41467-023-43896-7

Figure Lengend Snippet: a Peritoneal cells were incubated for 6 h with DGNS and DGNS-FITC and the identity of macrophages versus non-macrophages was assessed by their expression of F4/80. b Intravenous injection of DGNS or DGNS-FITC into the tail of Apoe KO mice and analysis of FITC fluorescence. Representative pictures and quantification of FITC fluorescence using ImageJ software ( n = 3). c Schematic of the generation of DGNS-Ctrl and DGNS- Zeb1 and ex vivo administration into macrophages. d Macrophages from both genotypes were assessed for cholesterol efflux as in Fig. . (APOAI n = 4,5,5,5; HDL n = 5,5,5,4). e As in d , but macrophages from both genotypes were assessed for BODIPY staining ( n = 4). Representative captures (scale bar: 20 μm) and quantification of four independent experiments. Graphs represent mean values ± SEM with two-tailed unpaired Student’s t test ( b ) or two-tailed unpaired Mann-Whitney ( d , e ). p ≤ 0.001 (***), p ≤ 0.01 (**) or p ≤ 0.05 (*) levels, or non-significant (ns) for values of p > 0.05. Raw data along with p values from for statistical analyses are included in the Source Data file.

Article Snippet: The development of the conditional Zeb1 flox allele mouse ( Zeb1 fl/fl , which is herein designated as Zeb1 WT ) was carried out at the Transgenesis Unit, which is jointly affiliated with the National Biotechnology Center (CNB) and the Severo Ochoa’s Molecular Biology Center-Autonomous University of Madrid (CBMSO-UAM) (Madrid, Spain), both centers belonging to the Spanish National Research Council (CSIC).

Techniques: Incubation, Expressing, Injection, Fluorescence, Software, Ex Vivo, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation

doi: 10.1038/s41467-023-43896-7

Figure Lengend Snippet: a Schematic and timeline of the injection of 50 μg/kg of DGNS-Ctrl or DGNS- Zeb1 in Zeb1 ∆M / Apoe KO mice. b As in a , H&E staining of sections of liver and kidney from Zeb1 ∆M / Apoe KO mice injected with DGNS-Ctrl. Scale bar: 20 μm. ( n = 2,3) c Representative pictures of aortic sinus sections Zeb1 ∆M / Apoe KO mice. stained for ZEB1 (HPA027524, 1/500) and LAMP2/MAC3 (clone M3/84, 1/50). ( n = 3). Scale bar: 50 μm. d Representative images of the en face ORO staining of aorta of Zeb1 ∆M / Apoe KO mice at the end of the protocol as in ( a ) and quantification of the plaque area by ImageJ ( n = 10,7). e Representative pictures of H&E staining of aortic sinus sections of Zeb1 ∆M / Apoe KO mice and quantification of the plaque area (n = 7,8). Scale bar: 200 μm. f As in d , but staining for ORO. Scale bar: 200 μm ( n = 6). g As in f , but in liver sections ( n = 6,5). h Data from eleven plaques (7 males; 4 females), six stable and five rupture, from the endarterectomies of patients in GSE41571 were assessed for ZEB1 mRNA expression, divided into two cohorts by the median of ZEB1 expression ( ZEB1 high versus ZEB1 low ), and examined for their association with stable or ruptured plaques. i As in h , heatmap of genes associated with plaque stability and rupture in stable and ruptured plaques. j Analyses of plaques from the endarterectomies of 39 male patients in GSE163154 —14 without intra-plaque hemorrhage (non-IPH) and 25 with plaque hemorrhage (IPH)—were assessed for ZEB1 mRNA expression divided in two cohorts by the median of ZEB1 expression ( ZEB1 high versus ZEB1 low ), and examined for their association with IPH. k The endarterectomies from 24 patients (21 males; 3 females) were assessed for ZEB1 mRNA levels by qRT-PCR and segregated in two cohorts depending on whether they expressed ZEB1 above ( ZEB1 high , 11 patients) or below ( ZEB1 low , 13 patients) the median. The clinical history of patients in both cohorts was analyzed to determine whether they had (symptomatic) or not (asymptomatic) a cardiovascular event. Graphs represent mean values ± SEM with two-tailed unpaired Mann-Whitney ( d , e , f ) or Fisher’s exact test ( h , j , k ). p ≤ 0.001 (***), p ≤ 0.01 (**) or p ≤ 0.05 (*) levels, or non-significant (ns) for values of p > 0.05. The raw data, along with p values from statistical analyses are included in the Source Data file.

Article Snippet: The development of the conditional Zeb1 flox allele mouse ( Zeb1 fl/fl , which is herein designated as Zeb1 WT ) was carried out at the Transgenesis Unit, which is jointly affiliated with the National Biotechnology Center (CNB) and the Severo Ochoa’s Molecular Biology Center-Autonomous University of Madrid (CBMSO-UAM) (Madrid, Spain), both centers belonging to the Spanish National Research Council (CSIC).

Techniques: Injection, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY