Journal: Oncotarget

Article Title: Pancreatic cancer-derived exosomes promote tumor metastasis and liver pre-metastatic niche formation

doi: 10.18632/oncotarget.18831

Figure Lengend Snippet: Confocal microscopy (lung, liver, spleen, brain) of PKH67-labeled Panc02 EXO and Panc02-H7 EXO tissue distribution (green) 24 hpi. (A) PKH-67-labeled liposomes served as controls (scale bar=100 μm). Histogram shows exosome tissue distribution quantification (n=5/group). CD45, p-Stat3, and CD11b IF staining in liver sections from controls (left) and mice treated with Panc02 EXOs(middle) or Panc02-H7 EXOs (right) for 12 d without tumor challenge. (B) Histogram shows infiltrating CD45 + cell quantification. FN and α-SMA IF staining in liver sections from controls (top) and mice treated with Panc02 EXOs(middle) or Panc02-H7 EXOs (bottom) for 12 d without tumor challenge. (C) Histogram shows infiltrating α-SMA + hStCs and FN expression quantification(400× magnification; n=5/group). Western blotting analysis showed upregulated S100A8 and S100A9 in livers treated with Panc02-H7-derived exosomes. Histogram shows expression of the three proteins in three groupsas determined by densitometric analysis (n=3/group). (D) Pancreatic cancer-derived exosomes induce MDSC accumulation in peripheral blood. (E) Representative flow cytometric plots (left) and quantification (right) of CD11b + GR1 + MDSCs (n=5/group). *P<0.05, **P<0.01,***P<0.001.

Article Snippet: Primary antibodies against fibronectin (1:100),α-SMA (1:50), S100A8 (1:50), S100A9 (1:50), and F4/80 (1:50) were purchased from Proteintech (Wuhan, China).

Techniques: Confocal Microscopy, Labeling, Liposomes, Staining, Expressing, Western Blot, Derivative Assay

Journal: Oncotarget

Article Title: Pancreatic cancer-derived exosomes promote tumor metastasis and liver pre-metastatic niche formation

doi: 10.18632/oncotarget.18831

Figure Lengend Snippet: IHC analysis and histopathological examination of macrophages (F4/80), hStCs (α-SMA), and neutrophils in liver metastatic niches of naïve mice and mice treated with PBS, Panc02 EXOs, or Panc02-H7 EXOs at 30d post-SOI (arrow shows neutrophils in liver) (A) . Representative histogram shows quantification of F4/80 + macrophages, α-SMA + hStCs, and neutrophils (B) . Identification of FN, S100A8, and S100A9 as inflammatory mediators, and collagen deposition in the liver metastatic niche (C) . Representative histogram shows FN and MTS quantification (D) . Representative histogram shows S100A8 and S100A9 quantification (E) . n=6/group.**P<0.01,***P<0.001.10 fields assessed per sample. FOV, field of view.

Article Snippet: Primary antibodies against fibronectin (1:100),α-SMA (1:50), S100A8 (1:50), S100A9 (1:50), and F4/80 (1:50) were purchased from Proteintech (Wuhan, China).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis

doi: 10.3390/ijms241210182

Figure Lengend Snippet: Comparison of mRNA and protein EPB41L3 levels in lung-tissue-derived fibroblasts between 14 IPF patients and 10 controls. Expression of EPB41L3 mRNA (normalized to that of β-actin) was measured using ( A ) RT-PCR, ( B ) densitometry, and ( C ) real-time PCR (log (2 −∆∆Ct ). Protein expression of EPB41L3 (normalized to that of β-actin) was measured using ( D ) Western blot and ( E ) densitometry. The Mann–Whitney U test was performed to identify the statistical significance between IPF and control groups. Data are medians and quantiles, and * p < 0.01. ( F ) Correlations between the RT-PCR and Western blot band intensities was analyzed using Spearman’s correlation coefficient. (See for RT-PCR and original Western blot images.)

Article Snippet: The membranes were blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: rabbit polyclonal anti-human EPB41L3 (1:2000; ProteinTech, Rosemont, IL, USA, #10719-I-AP), mouse monoclonal anti-human E-cadherin (1:1000; Invitrogen, #33-4000), mouse monoclonal anti-human N-cadherin (1:1000, Invitrogen, #33-3300), rabbit polyclonal anti-human collagen I (1:1000; Abcam, Cambridge, MA, USA, #MA1-26771), mouse monoclonal anti-human-α-SMA (1:500; Abcam, #ab7817), mouse monoclonal anti-human fibronectin (1:1000; Abcam, #ab6328), and mouse monoclonal anti-human β-actin (1:50,000; Sigma-Aldrich, St. Louis, MO, USA, #A1978).

Techniques: Comparison, Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, MANN-WHITNEY, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis

doi: 10.3390/ijms241210182

Figure Lengend Snippet: Changes in mRNA and protein of EPB41L3 and EMT-related genes expressed by A549 cells after stimulation with TGF-β. A549 cells were cultured with or without 5 ng/mL of TGF-β1 for 24, 48, and 72 h. The EPB41L3 -, N-cadherin -, E-cadherin -, and COL1A1 -to-β-actin ratios were measured using ( A ) real-time PCR, ( B ) Western blot, and ( C ) densitometry of protein bands. They were normalized to β-actin and expressed as ratios. All experiments were performed separately six times, and the representative images are shown. Data are mean ± SE of the six independent experiments. Basal levels (0 h) were excluded from statistical analysis. Significant differences between groups were evaluated using two-way ANOVA with Bonferroni multiple comparisons test. * p < 0.05 and ** p < 0.01 compared to TGF-β1-stimulated cells. Black bars: untreated control group, bars with diagonals: TGF-β1-treated group. (See for original Western blot images).

Article Snippet: The membranes were blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: rabbit polyclonal anti-human EPB41L3 (1:2000; ProteinTech, Rosemont, IL, USA, #10719-I-AP), mouse monoclonal anti-human E-cadherin (1:1000; Invitrogen, #33-4000), mouse monoclonal anti-human N-cadherin (1:1000, Invitrogen, #33-3300), rabbit polyclonal anti-human collagen I (1:1000; Abcam, Cambridge, MA, USA, #MA1-26771), mouse monoclonal anti-human-α-SMA (1:500; Abcam, #ab7817), mouse monoclonal anti-human fibronectin (1:1000; Abcam, #ab6328), and mouse monoclonal anti-human β-actin (1:50,000; Sigma-Aldrich, St. Louis, MO, USA, #A1978).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis

doi: 10.3390/ijms241210182

Figure Lengend Snippet: Effect of EPB41L3 transfection on EMT-related genes in the A549 cell line. Changes in EPB41L3 and EMT-related gene expression after EPB41L3 transfection in the presence and absence of 5 ng/mL of TGF-β1 for 48 h. Expression of mRNA and protein normalized to β-actin levels were measured using ( A-1 – A-4 ) real-time PCR, ( B ) Western blot, and ( C-1 – C-4 ) densitometry of protein bands. They were normalized to β-actin and expressed as ratios. Independent experiments were analyzed using densitometry. All experiments were performed separately six times, and the representative images are shown. Data are mean ± SE of 6 independent experiments. Significant differences between groups were evaluated using two-way ANOVA with Bonferroni multiple comparisons test. * p < 0.05 and ** p < 0.01. EMT: epithelial–mesenchymal transition, Lv: Lentivirus, black bar: control group, open bar: EPB41L3-overexpressing group. (See for original Western blot images).

Article Snippet: The membranes were blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: rabbit polyclonal anti-human EPB41L3 (1:2000; ProteinTech, Rosemont, IL, USA, #10719-I-AP), mouse monoclonal anti-human E-cadherin (1:1000; Invitrogen, #33-4000), mouse monoclonal anti-human N-cadherin (1:1000, Invitrogen, #33-3300), rabbit polyclonal anti-human collagen I (1:1000; Abcam, Cambridge, MA, USA, #MA1-26771), mouse monoclonal anti-human-α-SMA (1:500; Abcam, #ab7817), mouse monoclonal anti-human fibronectin (1:1000; Abcam, #ab6328), and mouse monoclonal anti-human β-actin (1:50,000; Sigma-Aldrich, St. Louis, MO, USA, #A1978).

Techniques: Transfection, Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis

doi: 10.3390/ijms241210182

Figure Lengend Snippet: Effect of silencing EPB41L3 on EMT-related genes in A549 cells. Changes of EPB41L3 expression in response to EPB41L3 siRNA in the presence and absence of 5 ng/mL of TGF-β1 for 48 h. Expression of mRNA and protein normalized to the β-actin level was measured using ( A ) real-time PCR, ( B ) Western blot, and ( C-1 – C-3 ) densitometry of protein bands. Independent experiments were analyzed using densitometry. They were normalized to β-actin and expressed as ratios. All experiments were performed separately six times, and the representative images are shown. Data are mean ± SE of 6 independent experiments. Significant differences between groups were evaluated using two-way ANOVA with Bonferroni multiple comparisons test. * p < 0.05. Black bar: control group, open bar: scramble group, Gray bar: EPB41L3-knockdown group. (See for original Western blot images).

Article Snippet: The membranes were blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: rabbit polyclonal anti-human EPB41L3 (1:2000; ProteinTech, Rosemont, IL, USA, #10719-I-AP), mouse monoclonal anti-human E-cadherin (1:1000; Invitrogen, #33-4000), mouse monoclonal anti-human N-cadherin (1:1000, Invitrogen, #33-3300), rabbit polyclonal anti-human collagen I (1:1000; Abcam, Cambridge, MA, USA, #MA1-26771), mouse monoclonal anti-human-α-SMA (1:500; Abcam, #ab7817), mouse monoclonal anti-human fibronectin (1:1000; Abcam, #ab6328), and mouse monoclonal anti-human β-actin (1:50,000; Sigma-Aldrich, St. Louis, MO, USA, #A1978).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis

doi: 10.3390/ijms241210182

Figure Lengend Snippet: Changes in mRNA and protein levels of EPB41L3 and FMT-related genes expressed by MRC5 cells after stimulation with TGF-β. MRC5 cells were cultured with or without 5 ng/mL of TGF-β1 for 24, 48, and 72 h. The EPB41L3 -, FN1 -, ACTA2 -, N-cadherin -, and COL1A1 -to-β-actin ratios were measured using ( A-1 – A-5 ) real-time PCR, ( B ) Western blot and ( C-1 – C-4 ) densitometry. Independent experiments were analyzed using densitometry. They were normalized to β-actin and expressed as ratios. All experiments were performed separately six times, and the representative images are shown. Data are mean ± SE of 6 independent experiments. Basal levels (0 h) were excluded from statistical analysis. Significant differences between groups were evaluated using two-way ANOVA with Bonferroni multiple comparisons test. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to TGF-β1-stimulated cells. Black bar: control group, open bar: EPB41L3-overexpressing group. (See for original Western blot images).

Article Snippet: The membranes were blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: rabbit polyclonal anti-human EPB41L3 (1:2000; ProteinTech, Rosemont, IL, USA, #10719-I-AP), mouse monoclonal anti-human E-cadherin (1:1000; Invitrogen, #33-4000), mouse monoclonal anti-human N-cadherin (1:1000, Invitrogen, #33-3300), rabbit polyclonal anti-human collagen I (1:1000; Abcam, Cambridge, MA, USA, #MA1-26771), mouse monoclonal anti-human-α-SMA (1:500; Abcam, #ab7817), mouse monoclonal anti-human fibronectin (1:1000; Abcam, #ab6328), and mouse monoclonal anti-human β-actin (1:50,000; Sigma-Aldrich, St. Louis, MO, USA, #A1978).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis

doi: 10.3390/ijms241210182

Figure Lengend Snippet: Effect of EPB41L3 transfection on FMT-related genes in the MRC5 cell line. Changes in the expression of EPB41L3 and FMT-related genes in response to EPB41L3 transfection in the presence and absence of 5 ng/mL of TGF-β1 for 48 h. Expression of mRNA and protein normalized to the β-actin level were measured using ( A-1 – A-3 ) real-time PCR, ( B ) Western blot, and ( C-1 – C-3 ) densitometry of protein bands. They were normalized to β-actin and expressed as ratios. Independent experiments were analyzed using densitometry. All experiments separately were performed six times, and the representative images are shown. Data are mean ± SE of 6 independent experiments. Significant differences between groups were evaluated using two-way ANOVA with Bonferroni multiple comparisons test. * p < 0.05 and ** p < 0.01. Lv: Lentivirus, black bar: control group, open bar: EPB41L3-overexpressing group. (See for original Western blot images).

Article Snippet: The membranes were blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: rabbit polyclonal anti-human EPB41L3 (1:2000; ProteinTech, Rosemont, IL, USA, #10719-I-AP), mouse monoclonal anti-human E-cadherin (1:1000; Invitrogen, #33-4000), mouse monoclonal anti-human N-cadherin (1:1000, Invitrogen, #33-3300), rabbit polyclonal anti-human collagen I (1:1000; Abcam, Cambridge, MA, USA, #MA1-26771), mouse monoclonal anti-human-α-SMA (1:500; Abcam, #ab7817), mouse monoclonal anti-human fibronectin (1:1000; Abcam, #ab6328), and mouse monoclonal anti-human β-actin (1:50,000; Sigma-Aldrich, St. Louis, MO, USA, #A1978).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis

doi: 10.3390/ijms241210182

Figure Lengend Snippet: Effect of silencing EPB41L3 on FMT-related genes in MRC5 cells. Changes in the expression of EPB41L3 and FMT-related genes in response to EPB41L3 siRNA. Expression of mRNA and protein were measured using ( A-1 – A-4 ) real-time PCR, ( B ) Western blot, and ( C-1 – C-4 ) densitometry of protein bands. Independent experiments were analyzed using densitometry. They were normalized to β-actin and expressed as ratios. All experiments separately were performed six times, and the representative images are shown. Data are mean ± SE of 6 independent experiments. Significant differences between groups were evaluated using two-way ANOVA with Bonferroni multiple comparisons test. * p < 0.05. Black bar: control group, open bar: scramble group, gray bar: EPB41L3-knockdown group. (See for original Western blot images).

Article Snippet: The membranes were blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: rabbit polyclonal anti-human EPB41L3 (1:2000; ProteinTech, Rosemont, IL, USA, #10719-I-AP), mouse monoclonal anti-human E-cadherin (1:1000; Invitrogen, #33-4000), mouse monoclonal anti-human N-cadherin (1:1000, Invitrogen, #33-3300), rabbit polyclonal anti-human collagen I (1:1000; Abcam, Cambridge, MA, USA, #MA1-26771), mouse monoclonal anti-human-α-SMA (1:500; Abcam, #ab7817), mouse monoclonal anti-human fibronectin (1:1000; Abcam, #ab6328), and mouse monoclonal anti-human β-actin (1:50,000; Sigma-Aldrich, St. Louis, MO, USA, #A1978).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Knockdown