Journal: Molecular Cancer

Article Title: MicroRNA-223 delivered by platelet-derived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3

doi: 10.1186/s12943-015-0327-z

Figure Lengend Snippet: Direct regulation of EPB41L3 expression by miR-223. (A) Schematic depicting the hypothetical duplexes formed by the interactions between the binding sites in the EPB41L3 3′-UTR (top) and miR-223 (bottom). The predicted free energy value of each hybrid is indicated. The seed recognition sites are denoted, and all nucleotides in these regions are highly conserved across species, including human, mouse and rat. (B) Quantitative RT-PCR analysis of miR-223 levels in A549 cells treated with pre-miR-control and pre-miR-223. (C) Firefly luciferase reporters containing wild-type (WT) or mutant (MUT) miR-223 binding sites in the EPB41L3 3′-UTR were co-transfected into A549 cells along with pre-miR-control, or pre-miR-223. he cells were assayed using a luciferase assay kit 24 h post-transfection. (D-E) Western blotting analysis of EPB41L3 protein levels in A549 cells treated with pre-miR-control, or pre-miR-223. D: representative image; E: quantitative analysis. (F-G) Western blotting analysis of EPB41L3 protein levels in A549 cells treated with pre-miR-control plus control plasmid, pre-miR-control plus EPB41L3 plasmid, pre-miR-223 plus control plasmid, or pre-miR-223 plus EPB41L3 plasmid. F: representative image; G: quantitative analysis. Data are presented as the mean ± SEM of five independent experiments (*, P < 0.05, **, P < 0.01,***, P < 0.001).

Article Snippet: A mammalian expression plasmid encoding the human EPB41L3 open reading frame (pReceiver-M02-EPB41L3) was purchased from GeneCopoeia (Germantown, MD).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Control, Luciferase, Mutagenesis, Transfection, Western Blot, Plasmid Preparation

Journal: Molecular Cancer

Article Title: MicroRNA-223 delivered by platelet-derived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3

doi: 10.1186/s12943-015-0327-z

Figure Lengend Snippet: Exogenous miR-223 delivered by P-MVs reduced EPB41L3 protein expression in A549 cells. (A-B) EPB41L3 protein levels of A549 cells treated without P-MVs (Mock), or with P-MVs from various sources for 24 h. A : representative image; B : quantitative analysis. Data are presented as the mean ± SEM of five independent experiments (*, P < 0.05, **, P < 0.01,***, P < 0.001).

Article Snippet: A mammalian expression plasmid encoding the human EPB41L3 open reading frame (pReceiver-M02-EPB41L3) was purchased from GeneCopoeia (Germantown, MD).

Techniques: Expressing

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 6. The chromatin-binding switch of O-GlcNAcylated FOXA1 is stimulated by MECP2. (A and B) O-GlcNAcylated FOXA1 chromatin loci shows transcriptional suppression characteristics. Average enrichment profiles of published MCF-7 cells (A) ATAC-seq (GSE179666), RNA-Pol II ChIP-seq (GSE54693), (B) H3K27me3 (GSE96363), H3K4me1 (GSE86714), H3K4me3 (GSE97481), and H3K27ac (GSE97481) ChIP-seq reads at differential quantitative FOXA1WT and FOXA13A ChIP-seq peaks are shown. (C) Heat maps of the FOXA1WT and FOXA13A ChIP-seq signal density at hg19 genome-wide CpG islands. (D) Average published MCF-7 cells genome-wide DNA methylation (GSE54693) profiles at CpG islands (left) and typical/super enhancer elements (right) of differential quantitative FOXA1WT and FOXA13A ChIP-seq peaks. (E) Heat map representation of endogenic MECP2 ChIP-seq (in FOXA1WT and FOXA13A expressed MCF-7 FOXA1 KO cells, respectively) signal enrichment at differential quantitative FOXA1WT and FOXA13A sites. (F) Cells were transfected with MECP2 shRNA (shMECP2). Average enrichment profiles of FOXA1 ChIP-seq reads at FOXA1WT and FOXA13A- biased sites are shown. (G) The box plots showing the mRNA expression changes of FOXA1-targeting genes associated with FOXA1WT and FOXA13A-biased sites. n = 2 biologically independent RNA-seq replicates. (H) Predicted regulatory network between FOXA1 and the corresponding downstream genes in different O-GlcNAcylation state. Functional enrichment is indicated. (I) Integrative Genomics Viewer tracks showing FOXA1, MECP2, RNA-Pol II ChIP-seq and DNA methylation, ATAC-seq signal at the promoter regions of the representative genes EPB41L3 and COL9A2. (J) Certain FOXA1 targeting genes were validated by qPCR and ChIP-qPCR. (K) Methylation status of EPB41L3 and COL9A2 gene promoter regions in the indicated cells were measured by methylation-specific qPCR. (L) Cells were transfected with MECP2 shRNA (shMECP2). Certain FOXA1 targeting genes were validated by qPCR and ChIP-qPCR. For (J) to (L), cells were pretreated with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours. n = 4 biologically independent experiments.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Binding Assay, ChIP-sequencing, Genome Wide, DNA Methylation Assay, Transfection, shRNA, Expressing, RNA Sequencing, Functional Assay, ChIP-qPCR, Methylation

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 7. FOXA1 O-GlcNAcylation elevates the metastatic potential of breast cancer cells. (A to C) The motility (A), migration and invasion ability (B), and adhesion ability (C) of breast cancer cells were analyzed. Cells were treated with OSMI-4 (50 μM) or TMG (50 μM) for 24 hours. Mitomycin C was used to inhibit the impact of cell proliferation. Representative and quantified results of the wound-healing (A; scale bar, 200 μm), Transwell (B, scale bar, 100 μm) and adhesion (C; scale bar, 100 μm) assays in FOXA1WT- or FOXA13A-expressing breast cancer cells are shown. (A and B) n = 3 biologically independent experiments. (C) n = 6 biologically independent experiments. (D and E) The migration and invasion abilities were analyzed and quantified by Transwell assays in FOXA1WT- or FOXA13A-expressing breast cancer cells (MCF-7 FOXA1 KO and MDA-MB-231; scale bar, 100 μm). Cells were transfected with MECP2 (D) or EPB41L3 (E) shRNA (shMECP2 and shEPB41L3) or scrambled shRNA (control) for 48 hours before the assays. Mitomycin C was used to inhibit the impact of cell proliferation. n = 3 biologically independent experiments. (F) FOXA1 immunoprecipitation was performed in five breast cancer patient tumor samples, and immunoprecipitated fractions were analyzed by WB. The pathological grade and the patient no. are indicated.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Migration, Expressing, Transfection, shRNA, Control, Immunoprecipitation

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 8. FOXA1 O-GlcNAcylation promotes oncogenesis and metastasis of breast cancer in vivo. (A and B) Elimination of FOXA1 O-GlcNAcylation diminished xe- nograft tumor formation in vivo. WT MCF-7, MCF-7 FOXA1 KO, FOXA1WT, or FOXA13A MCF-7 FOXA1 KO cells were injected subcutaneously into the axillae of nude mice (n = 5 for each group). Mice were euthanized after 24 days, and their tumor masses were excised (A), measured and weighed (B). (C) HA-FOXA1 immunoprecipitation was performed with anti-HA-tag magnetic beads using xenograft tumor samples in each group, and the immunoprecipitated fractions were analyzed by WB. (D) Xenograft tumor samples were subjected to H&E, EPB41L3, and Ki67 staining. One representative experiment of n = 3 independent experiments is shown. Scale bar, 100 μm. (E) Schematics for tail vein injection of breast cancer cells in nude mice to generate experimental pulmonary metastasis. (F) MCF-7, MCF-7 FOXA1 KO, FOXA1WT, or FOXA13A

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: In Vivo, Injection, Immunoprecipitation, Magnetic Beads, Staining

Journal: Science advances

Article Title: FOXA1 O-GlcNAcylation-mediated transcriptional switch governs metastasis capacity in breast cancer.

doi: 10.1126/sciadv.adg7112

Figure Lengend Snippet: Fig. 9. Schematic working model of FOXA1 O-GlcNAcylation–mediated breast cancer metastasis. O-GlcNAcylation at T432/S441/S443 shapes the FOXA1 interac- tome, especially triggers the recruitment of transcriptional repressor MECP2, consequently stimulates FOXA1 chromatin-binding sites switch to chromatin loci of adhe- sion-related genes, including EPB41L3 and COL9A2, and thus promotes breast cancer proliferation and metastasis both in vitro and in vivo.

Article Snippet: Lentiviral shRNA plasmids for FOXA1 (#sc-37930-V), MECP2 (#sc35892-V, #sc-156056-V), and EPB41L3 (#sc-40291-V) were purchased from Santa Cruz Biotechnology. shRNA plasmids for EPB41L3 (#TL313194V) and FOXA1 (#TL312942V) were purchased from OriGene Technologies.

Techniques: Binding Assay, In Vitro, In Vivo

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: Figure 1. 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 Figure S1 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: Figure 2. 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 Figure S2 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: Figure 3. 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 represen- tative 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 Figure S3 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: Figure 4. 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 Figure S4 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: Figure 5. 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 Figure S5 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: Figure 6. 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 Figure S6 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: Figure 7. Effect of silencing EPB41L3 on FMT-related genes in MRC5 cells. Changes in the ex- pression 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) den- sitometry 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 Figure S7 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