Journal: Science Advances

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1126/sciadv.adn6068

Figure Lengend Snippet: ( A ) Masson trichrome staining (MTS) in kidneys of male CD-1 mice. Scale bar, 50 μm. ( B ) Immunofluorescence analysis of vimentin/E-cadherin and αSMA/CD31 in kidneys from nondiabetic and diabetic mice. FITC-labeled E-cadherin, rhodamine-labeled vimentin, and DAPI (nuclei, blue); FITC-labeled αSMA, rhodamine-labeled CD31, and DAPI (nuclei, blue). Representative merged (original magnification 400×) images are shown. Scale bar, 50 μm. ( C ) mRNA array analysis in control and diabetic mice. Heatmap of analyzed gene expression. ( D ) Relative gene expression levels of Angptl4 , Angplt3 , and fibronectin in control and diabetic mice. n = 7 mice per group. ( E and F ) Physiological characteristics in diabetic and nondiabetic mice of both genotypes. n = 7 mice per group except for blood pressure where n = 6 mice per group. Two replicate experiments were analyzed. Data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ( G ) Histologic images of MTS, Sirius red, and PAS staining in diabetic control and Angptl4 −/− mice (original magnification, 300×). Quantification was performed using ImageJ. Representative images are shown. Scale bar, 100 μm. n = 7 mice per group. ( H ) Immunofluorescence analysis of collagen I, Vimentin, and αSMA/CD31 was performed in kidneys from diabetic control and diabetic Angptl4 −/− mice. FITC-labeled collagen I, rhodamine-labeled vimentin, FITC-labeled αSMA, rhodamine-labeled E-cadherin, and DAPI (nuclei, blue) were used. Representative merged (original magnification, 200×) images are shown. Scale bar, 50 μm. n = 7 mice per group. Three independent experiments were analyzed. ( I ) Plasma levels of IL-1β, IL-5, IL-6, CCL3, CCL5, eotaxin, and Mo-GCSF were analyzed by cytokine array analysis. n = 3 to 5 control mice and n = 5 Angptl4 −/− mice. Data are mean ± SEM. Student’s t test (unpaired two tailed) was used for analysis of statistical significance. Significance: * P < 0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (no. 40-9800) was from Invitrogen, and mouse monoclonal anti-αSMA (catalog no. A5228) and mouse monoclonal anti–β-actin (AC-74) (A2228) antibodies were from Sigma (St. Louis, MO).

Techniques: Staining, Immunofluorescence, Labeling, Control, Expressing, Comparison, Two Tailed Test

Journal: Science Advances

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1126/sciadv.adn6068

Figure Lengend Snippet: ( A to C ) Immunohistochemical analysis of CPT1a, PGC1α, SIRT3, PKM2, PDK4, HIF1α, and Snail1 expression in kidneys from nondiabetic and diabetic, control, and Angptl4 −/− mice. Scale bar, 50 μm. n = 6 mice per group. Two independent replicate experiments were performed. Representative micrographic images (original magnification, 300×) are shown. ( D ) Radiolabeled [ 14 C]palmitate oxidation and [ 14 CO 2 ] release were measured. CPM of each sample was counted. n = 5 per group. ( E ) Fatty acid oxidation analysis in isolated tubules, podocytes, and endothelial cells from nondiabetic and diabetic, control, and Angptl4 −/− mice. Radiolabeled [ 14 C]palmitate oxidation and [ 14 CO 2 ] release were measured. CPM of each sample was counted ( n = 5 per group). Data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. Significance: * P < 0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (no. 40-9800) was from Invitrogen, and mouse monoclonal anti-αSMA (catalog no. A5228) and mouse monoclonal anti–β-actin (AC-74) (A2228) antibodies were from Sigma (St. Louis, MO).

Techniques: Immunohistochemical staining, Expressing, Control, Isolation, Comparison

Journal: Science Advances

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1126/sciadv.adn6068

Figure Lengend Snippet: ( A ) Proximity between Angptl4 and β-integrin in HK-2 cells and ( B ) podocytes in cultured HK-2 cells treated with TGFβ1 (10 ng/ml) for 48 hours. Images at 400× original magnification were obtained from six different areas. Scale bar, 50 μm. ( C ) Proximity between DPP-4 and β-integrin in tubules cocultured with podocytes from the indicated mice. Images at 400× original magnification, were obtained from six different areas. Scale bar, 50 μm. ( D ) Proximity between DPP-4 and β-integrin in podocytes cocultured with tubules from the indicated mice. For each slide, images at 400× original magnification were obtained from six different areas. Scale bar, 50 μm. ( E ) Representative MTS and Sirius red staining in kidneys of control and linagliptin-treated diabetic mice. Scale bar, 50 μm. n = 6 mice per group. ( F ) Representative MTS and Sirius red staining in kidneys from IgG control-treated and N–Integrin β1–treated diabetic mice. Scale bar, 50 μm. n = 6 mice per group. ( G ) Data from www.mirbase.org suggest miR-29a nucleotide sequence alignment with the 3′UTR of Angptl4 mRNA. ( H ) Angptl4 3′UTR transcriptional activity measurement in the presence of antagomirs or ( I ) mimics of microRNA 29a, b, and c. Two independent experiments were analyzed. ( J ) microRNA array analysis of miR-29 family members in control and diabetic mice. ( K ) Representative images of MTS in kidneys after LNA–miR-29 treatment in control and diabetic mice. Scale bar, 50 μm. n = 7 mice per group. ( L ) Representative images of MTS in kidneys after LNA–miR-29 treatment in control and unilateral urinary obstruction (UUO)–operated mice. Scale bar, 50 μm. n = 5 mice per group. ( M ) Radiolabeled [ 14 C]palmitate oxidation and [ 14 C]CO 2 uptake were measured in indicated groups. CPM of each sample was counted. n = 6 per group. ( N ) Angplt4 mRNA expression in the indicated groups. n = 6 per group. Data are mean ± SEM. One-way ANOVA was used for the analysis of statistical significance. Significance: * P < 0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (no. 40-9800) was from Invitrogen, and mouse monoclonal anti-αSMA (catalog no. A5228) and mouse monoclonal anti–β-actin (AC-74) (A2228) antibodies were from Sigma (St. Louis, MO).

Techniques: Cell Culture, Staining, Control, Sequencing, Activity Assay, Expressing

Journal: Science Advances

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1126/sciadv.adn6068

Figure Lengend Snippet: ( A ) Schematic diagram showing the treatment protocol of control ASO and kidney-specific Angplt4 ASO treatment in diabetic and nondiabetic C57BL/6 mice. Five doses of STZ (50 mg kg −1 day −1 ip) were injected to induce diabetes; after 16 weeks, the mice received either control ASO or Angptl4 ASO for 8 weeks. Art was created using Servier medical art illustrations. ( B ) RT-qPCR gene expression analysis of Angptl4 in kidneys of nondiabetic and diabetic, and control ASO- and Angplt4 ASO-treated mice. n = 7 per group. ( C to G ) Physiological parameters of nondiabetic and diabetic, and control ASO- and Angplt4 ASO-treated mice. n = 8 mice per group, combined from two replicate experiments. ( H ) Representative histologic images from MTS, Sirius red, and PAS staining in kidneys from nondiabetic and diabetic, and control ASO- and Angplt4 ASO-treated mice (original magnification, 300×). Relative area of fibrosis (%) and relative collagen (%) were quantified using ImageJ. Scale bar, 50 μm. ( I ) Representative transmission electron microscopy images are shown. n = 3 per group. Scale bar, 1 μm. Relative density of podocyte foot processes and GBM thickness were calculated using ImageJ software. Six independent images of the staining were analyzed. ( J ) Immunofluorescence analysis of αSMA/E-cadherin and αSMA/CD31 in kidneys of ASO treatment in control and diabetic mice. FITC-labeled E-cadherin, rhodamine-labeled αSMA, and DAPI (nuclei, blue), and FITC-labeled αSMA, rhodamine-labeled CD31, and DAPI (nuclei, blue) were used. Representative merged (original magnification, 400×) images are shown. Scale bar, 50 μm. n = 8 mice per group. Three independent experiments were analyzed. Data are mean ± SEM. One-way ANOVA was used for the analysis of statistical significance. Significance: * P < 0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (no. 40-9800) was from Invitrogen, and mouse monoclonal anti-αSMA (catalog no. A5228) and mouse monoclonal anti–β-actin (AC-74) (A2228) antibodies were from Sigma (St. Louis, MO).

Techniques: Control, Injection, Quantitative RT-PCR, Expressing, Staining, Transmission Assay, Electron Microscopy, Software, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1101/2023.11.08.565844

Figure Lengend Snippet: A. Masson trichrome staining in kidneys of CD-1 mice at 16-weeks and 24-weeks post streptozotocin injection. Scale bar 50 μm. B. Immunofluorescence analysis of vimentin/E-cadherin and αSMA/CD31 in kidneys from nondiabetic (control) and diabetic mice. FITC-labeled E-cadherin, rhodamine-labeled vimentin and DAPI (nuclei, blue); FITC-labeled αSMA, rhodamine-labeled CD31 and DAPI (nuclei, blue), were used. Representative merged (original magnification 400X) images are shown. Scale bar 50 μm.. C. mRNA array analysis in control and diabetic mice. Heat map of analyzed gene expression is shown. D. Relative gene expression levels of Angptl4 , Angplt3 , and fibronectin in control and diabetic mice. n=7 mice/group. E. Immunohistochemical images of Angptl4 expression in human kidney tissue from https://www.proteinatlas.org . F. Physiological characteristics in nondiabetic and diabetic control and Angptl4 -/- mice. n=7 mice/group except for blood pressure where n=6 mice/group. Two replicate experiments were analyzed. G. Histologic images of Masson trichrome, Sirius red and PAS staining in diabetic control and Angptl4 -/- mice (original magnification 300X). Relative area of fibrosis, relative collagen deposition and glomerular surface area were calculated using ImageJ. Representative images are shown. Scale bar 100 μm. n=7 mice/group. H. Immunofluorescence analysis of collagen I and αSMA/CD31 was performed in kidneys from diabetic control and diabetic Angptl4 -/- mice. FITC-labeled collagen I, FITC-labeled αSMA, rhodamine-labeled E-cadherin and DAPI (nuclei, blue), were used. Representative merged (original magnification 200X) images are shown. Scale bar 50 μm. n=7 mice/group. Three independent experiments were analyzed. I. Plasma levels of IL-1β, IL-5, IL-6, CCL3, CCL5, eotaxin and Mo-GCSF from diabetic control and diabetic Angptl4 -/- mice were analyzed by cytokine array analysis. n=5 mice/group. Data are mean ± SEM. One-way Anova with Tukey’s multiple comparison test was used to calculate statistical significance. Significance-* p <0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (#40-9800) was from Invitrogen, mouse monoclonal anti-αSMA (Cat: A5228) and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were from Sigma (St Louis, MO).

Techniques: Staining, Injection, Immunofluorescence, Labeling, Expressing, Immunohistochemistry, Comparison

Journal: bioRxiv

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1101/2023.11.08.565844

Figure Lengend Snippet: A-C. Immunohistochemical analysis of, CPT1a, PGC1α, SIRT3, PKM2, PDK4, HIF1α and Snail1 expression in kidneys from nondiabetic and diabetic, control and Angptl4 -/- mice. Scale bar 50 μm. n=6 mice/group. Two independent replicate experiments were performed. Representative micrographic images (original magnification 300X) are shown. D. Radiolabeled [ 14 C]palmitate oxidation was measured and and [ 14 CO 2 ] release were measured. CPM of each sample was counted. n=5/group. E. Fatty acid oxidation analysis in isolated tubules, podocytes and endothelial cells from nondiabetic and diabetic control and Angptl4 -/- mice. Radiolabeled [ 14 C]palmitate oxidation and [ 14 CO 2 ] release were measured. CPM of each sample was counted (n=5/group). Data are mean ± SEM. One-way Anova with Tukey’s multiple comparison test was used to calculate statistical significance. Significance-* p <0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (#40-9800) was from Invitrogen, mouse monoclonal anti-αSMA (Cat: A5228) and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were from Sigma (St Louis, MO).

Techniques: Immunohistochemistry, Expressing, Isolation, Comparison

Journal: bioRxiv

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1101/2023.11.08.565844

Figure Lengend Snippet: A. Schematic diagram, showing induction of diabetes in Angptl4 fl/fl; Podocin Cre- (Control) and in Angptl4 fl/fl; podocin Cre+ (pmut) mice. Five doses of STZ (50 mg/kg/day IP) were injected to induce diabetes in mice; after 16 weeks kidneys were evaluated for fibrosis. Art was created using Servier medical art illustrations. B. Physiological parameters in diabetic and non-diabetic mice of both genotypes. n=6 mice/group combined from two replicate experiments were evaluated. C. Histologic images from Sirius red and PAS staining from kidneys of non-diabetic and diabetic control and pmut mice. Representative images (original magnification 300X) are shown. Relative area of fibrosis (%) and relative collagen (%) were measured using the ImageJ program. Scale bar 50 µm. D. Representative transmission electron microscopy images are shown. n=3/group. Scale bar 1 μm. Relative density of podocyte foot processes and glomerular basement membrane (GBM) thickness were calculated using ImageJ software. Six independent images of the staining were analyzed. E. After 48 h, albumin was measured in the cell media of cultured isolated podocytes from nondiabetic control and pmut, and diabetic control and pmut mice. F. qPCR mRNA gene expression of α SMA and FSP-1 in isolated podocytes from nondiabetic control and pmut and diabetic control and pmut mice. n=4/non-diabetic group and n=5/diabetic group. G. IL-1β, IL-6, and TNFα levels measured in culture media. H. Radiolabeled [C ]palmitate uptake analysis and I. [ C] palmitate oxidation in isolated podocytes from indicated groups. CPM of each sample was counted. J. ATP measurement via calorimetric assay in isolated podocytes. K. Schematic diagram showing co-culture experimental set-up. Isolated podocytes from diabetic control and diabetic pmut mice were cocultured with isolated tubules from control mice. L. Representative Duolink In Situ images of TGF-βR1/2 from tubules that were co-cultured with podocytes either from diabetic control or diabetic pmut mice and corresponding quantification. Combination sites of TGF-βR1/2 in cells were counted in 10 different areas per condition (original magnification 400X). M. qPCR gene expression analysis of FSP-1 , α SMA and TGF β R1. N. Measurement of IL-1β, IL-6, IL-10, IL-17 and TNFα levels in the cell supernatant. O. Radiolabeled [ C]palmitate uptake and oxidation, were measured in indicated groups. CPM of each sample was counted. P. ATP measurement in isolated tubules Q. qPCR gene expression analysis of miR-29a-3p, miR-29b-3p and miR-29c-3p in the tubules cocultured with podocytes from diabetic control and diabetic pmut mice. Data are mean ± SEM. One-way Anova with Tukey’s multiple comparison test was used to calculate statistical significance. Significance-* p <0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (#40-9800) was from Invitrogen, mouse monoclonal anti-αSMA (Cat: A5228) and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were from Sigma (St Louis, MO).

Techniques: Injection, Staining, Transmission Assay, Electron Microscopy, Membrane, Software, Cell Culture, Isolation, Expressing, Co-Culture Assay, In Situ, Comparison

Journal: bioRxiv

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1101/2023.11.08.565844

Figure Lengend Snippet: A. Schematic diagram, showing induction of diabetes in Angptl4 fl/fl; iPAX8 Cre- (Control) and in doxycycline-treated Angptl4 fl/fl; podocin iPax8 Cre+ (tmut). Five doses of STZ (50 mg/kg/day IP) were injected to induce diabetes; after 16 weeks kidneys were evaluated for analysis of fibrosis. Art was created using Servier medical art illustrations. B. Physiological parameters were measured in diabetic and non-diabetic mice of both genotypes. n=5 mice/group combined from two replicate experiments. C. Representative histologic images of Sirius red and PAS staining in kidneys from non-diabetic and diabetic control and tmut mice. (Original magnification 300X). Relative area of fibrosis (%) and relative collagen (%) were measured using ImageJ. Scale bar 50 µm. C. Representative transmission electron microscopy images. n=3/group. Scale bar 1 μm. Relative density of podocyte foot processes and glomerular basement membrane (GBM) thickness were calculated using ImageJ software. Six independent images of the staining were analyzed. E. qPCR gene expression of α SMA and FSP-1 in tubules from nondiabetic control and tmut and diabetic control and tmut mice. n=5/group. F. IL-1β and IL-6 measurement in the media of cultured tubules from nondiabetic control and tmut and diabetic control and tmut mice. n=5/group. G. Radiolabeled [C ]palmitate uptake analysis and radiolabeled [ C]palmitate oxidation were measured in tubules from the indicated groups. CPM of each sample was counted. H. ATP measurement in the cultured tubules from nondiabetic control and tmut and diabetic control and tmut mice. I. Schematic diagram showing co-culture experimental set-up. Isolated tubules from diabetic control and diabetic tmut mice were cocultured with podocytes from control mice. J. After 48 h, albumin was measured in the cell media of podocytes co-cultured either with tubules from diabetic control and tmut mice. K. qPCR gene expression analysis of FSP-1 , α SMA and TGF β R1 in the podocytes cocultured either with tubules from diabetic control or diabetic tmut mice L. Representative Duolink In Situ images of TGF-βR1/2 from podocytes that were co-cultured with tubules either from diabetic control or diabetic tmut mice and corresponding quantification. Combination sites of TGF-βR1/2 in cells were counted in 10 different areas per condition (original magnification 400X). M. Measurement of IL-1β, IL-6, and TNFα levels in the cell supernatant. N. Radiolabeled [ C]palmitate uptake and oxidation, were measured in indicated groups. CPM of each sample was counted. O. ATP measurement in the podocytes which were co-cultured with tubules either from diabetic control or diabetic tmut mice. P. qPCR gene expression analysis of miR-29a-3p, miR-29b-3p and miR-29c-3p in podocytes which were cocultured with tubules from diabetic control and diabetic tmut mice. Data are mean ± SEM. One-way Anova with Tukey’s multiple comparison test was used to calculate statistical significance. Significance-* p <0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (#40-9800) was from Invitrogen, mouse monoclonal anti-αSMA (Cat: A5228) and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were from Sigma (St Louis, MO).

Techniques: Injection, Staining, Transmission Assay, Electron Microscopy, Membrane, Software, Expressing, Cell Culture, Co-Culture Assay, Isolation, In Situ, Comparison

Journal: bioRxiv

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1101/2023.11.08.565844

Figure Lengend Snippet: A. Proximity between Angptl4 and β-integrin was assessed by DuoLink In Situ Assay in cultured HK-2 cells treated with TGFβ1 (10 ng/ml) for 48 h. For each slide, images at 400X original magnifications were obtained from six different areas. Scale bar 50 µm. B. Proximity between Angptl4 and β-integrin was assessed by DuoLink In Situ Assay in immortalized podocytes treated with TGFβ1 (10 ng/ml) for 48 h. For each slide, images at 400X original magnification were obtained from six different areas. Scale bar 50 µm C. Proximity between DPP-4 and β-integrin was assessed by DuoLink In Situ Assay in tubules co-cultured either with podocytes from diabetic control or diabetic pmut mice. For each slide, images at 400X original magnification were obtained from six different areas. Scale bar 50 µm. D. Proximity between DPP-4 and β-integrin was assessed by DuoLink In Situ Assay in podocytes co-cultured either with tubules from diabetic control or diabetic tmut mice. For each slide, images at 400X original magnification were obtained from six different areas. Scale bar 50 µm. E. Representative Masson trichrome and Sirius red staining in kidneys of control and linagliptin-treated diabetic mice. Linagliptin was administered for 8 weeks. Scale bar 50 µm. n=6 mice/group. F. Representative Masson trichrome and Sirius red staining in kidneys from IgG control-treated and N-integrin-β1-treated diabetic mice IgG and N-integrin-β1 were provided to diabetic mice for 4 weeks. Scale bar 50 µm. n=6 mice/group. G. Data from microRNA.org suggests miR-29a nucleotide sequence alignment with the 3’untranslated region of Angptl4 mRNA. H. Angptl4 3’UTR transcriptional activity measurement in the presence of antagomirs for microRNA 29a, b and c. I. Angptl4 3’UTR transcriptional activity measurement in the presence of mimics of microRNA 29a, b and c. Two independent experiments were analyzed. J. microRNA array analysis of miR-29 family members in control and diabetic mice. K. Representative images of Masson trichrome staining of kidneys after LNA-miR-29 treatment in control and diabetic mice. Scale bar 50 µm. n=7 mice/group. L. Representative images of Masson trichrome staining in kidneys after LNA-miR-29 treatment in control and unilateral urinary obstruction (UUO)-operated mice. Scale bar 50 µm. n=5 mice/group. M. Radiolabeled [ 14 C]palmitate oxidation and [ 14 C]CO 2 ptake were measured in indicated groups. CPM of each sample was counted. n=6/group. Data are mean ± SEM. One-way ANOVA was used for the analysis of statistical significance. Significance-* p <0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (#40-9800) was from Invitrogen, mouse monoclonal anti-αSMA (Cat: A5228) and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were from Sigma (St Louis, MO).

Techniques: In Situ, Cell Culture, Staining, Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease

doi: 10.1101/2023.11.08.565844

Figure Lengend Snippet: A. Schematic diagram showing the treatment protocol of control ASO and kidney-specific Angplt4 ASO treatment in diabetic and non-diabetic C57BL/6 mice. Five doses of STZ (50 mg/kg/day IP) were injected to induce diabetes; after 16 weeks the mice received either control ASO or Angptl4 ASO for eight weeks. Art was created using Servier medical art illustrations. B. RT-qPCR gene expression analysis of Angptl4 in kidneys of nondiabetic and diabetic, control ASO-and Angplt4 ASO-treated mice. n=7/group. C-G. Physiological parameters of nondiabetic and diabetic, control ASO-and Angplt4 ASO-treated mice. n=8 mice/group, combined from two replicate experiments. H. Representative histologic images from MTS, Sirius red and PAS staining in kidneys from nondiabetic and diabetic, control ASO-and Angplt4 ASO-treated mice (original magnification 300X). Relative area of fibrosis (%) and relative collagen (%) were quantified using ImageJ. Scale bar 50 µm. I. Representative transmission electron microscopy images are shown. n=3/group. Scale bar 1 μm. Relative density of podocyte foot processes and glomerular basement membrane (GBM) thickness were calculated using ImageJ software. Six independent images of the staining were analyzed. J. Immunofluorescence analysis of αSMA/E-cadherin and αSMA/CD31 in kidneys of ASO treatment in control and diabetic mice. FITC-labeled E-cadherin, rhodamine-labeled αSMA and DAPI (nuclei, blue); FITC-labeled αSMA, rhodamine-labeled CD31 and DAPI (nuclei, blue), were used. Representative merged (original magnification 400X) images are shown. Scale bar 50 μm. n=8 mice/group. Three independent experiments were analyzed. Data are mean ± SEM. One-way ANOVA was used for the analysis of statistical significance. Significance-* p <0.05.

Article Snippet: Rabbit polyclonal antibody against ANGPTL4 (#40-9800) was from Invitrogen, mouse monoclonal anti-αSMA (Cat: A5228) and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were from Sigma (St Louis, MO).

Techniques: Injection, Quantitative RT-PCR, Expressing, Staining, Transmission Assay, Electron Microscopy, Membrane, Software, Immunofluorescence, Labeling

Journal: Oncology Letters

Article Title: Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

doi: 10.3892/ol.2022.13419

Figure Lengend Snippet: Immunohistochemical staining of ANGPTL4. Immunohisto-chemical staining for cancer tissues of patients with TC was performed using an anti-ANGPTL4 antibody. Representative images showing TC cells that were found to exhibit (A) a high (positive cell rate >30%) or a low [positive cell rate (B) 1-30% or (C) ≈0%) rate of ANGPTL4 expression. (D) Noncancerous tongue epithelial cells. Scale bars, 100 µm. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Article Snippet: Sections were then incubated (4°C overnight) with a rabbit polyclonal antibody to ANGPTL4 (20 μg/m1; ab196746, Abcam) and stained at room temperature for 20 min using EnVision+ solution (Dako Cytomation) and 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.006% H 2 O 2 , according to the manufacturer's instructions.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncology Letters

Article Title: Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

doi: 10.3892/ol.2022.13419

Figure Lengend Snippet: Association between cancer-cell ANGPTL4 expression and patient clinicopathological parameters in tongue cancer.

Article Snippet: Sections were then incubated (4°C overnight) with a rabbit polyclonal antibody to ANGPTL4 (20 μg/m1; ab196746, Abcam) and stained at room temperature for 20 min using EnVision+ solution (Dako Cytomation) and 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.006% H 2 O 2 , according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

doi: 10.3892/ol.2022.13419

Figure Lengend Snippet: Association of overall patient survival rate with ANGPTL4 expression in TC. The overall survival rate was evaluated for groups of patients with TC classified by high (n=15; solid line) or low (n=33; dashed line) rates of ANGPTL4 expression in their collected TC tissue samples. Statistical analysis was performed using the Kaplan-Meier method and verified using the log-rank test. The table of numbers at risk at the bottom of the figure indicates the number of patients living just before the time point. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Article Snippet: Sections were then incubated (4°C overnight) with a rabbit polyclonal antibody to ANGPTL4 (20 μg/m1; ab196746, Abcam) and stained at room temperature for 20 min using EnVision+ solution (Dako Cytomation) and 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.006% H 2 O 2 , according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

doi: 10.3892/ol.2022.13419

Figure Lengend Snippet: Univariate and multivariate analysis of overall survival in 48 patients with tongue cancer.

Article Snippet: Sections were then incubated (4°C overnight) with a rabbit polyclonal antibody to ANGPTL4 (20 μg/m1; ab196746, Abcam) and stained at room temperature for 20 min using EnVision+ solution (Dako Cytomation) and 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.006% H 2 O 2 , according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

doi: 10.3892/ol.2022.13419

Figure Lengend Snippet: ANGPTL4 plasma concentrations in patients with TC with or without lung metastasis. ANGPTL4 plasma concentrations were compared between patients with TC with and without lung metastasis. Open circles indicate individual ANGPTL4 plasma concentrations of patients with TC (n=20 each). Closed circles and bars denote averages and standard deviation values, respectively. Statistical analysis was performed using an unpaired Student's t-test. *P<0.001. The dashed line indicates the optimal cut-point of the plasma ANGPTL4 concentration for prediction of TC lung metastasis. The optimal cut-point was determined by bootstrapped receiver operating characteristic analysis under Liu's method using 1,000 bootstrap samples. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Article Snippet: Sections were then incubated (4°C overnight) with a rabbit polyclonal antibody to ANGPTL4 (20 μg/m1; ab196746, Abcam) and stained at room temperature for 20 min using EnVision+ solution (Dako Cytomation) and 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.006% H 2 O 2 , according to the manufacturer's instructions.

Techniques: Clinical Proteomics, Standard Deviation, Concentration Assay

Journal: Oncology Letters

Article Title: Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

doi: 10.3892/ol.2022.13419

Figure Lengend Snippet: Correlation of overall patient survival rate with plasma ANGPTL4 concentration in TC. The overall survival rate was evaluated for groups of patients with TC classified by ANGPTL4 concentration >9.1 ng/ml (n=20; solid line) or ≤9.1 ng/ml (n=20; dashed line). Statistical analysis was performed using the Kaplan-Meier method and verified using the log-rank test. The table of numbers at risk at the bottom of the figure indicates the number of patients living just before the time point. ANGPTL4, angiopietin-like 4; TC, tongue cancer.

Article Snippet: Sections were then incubated (4°C overnight) with a rabbit polyclonal antibody to ANGPTL4 (20 μg/m1; ab196746, Abcam) and stained at room temperature for 20 min using EnVision+ solution (Dako Cytomation) and 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.006% H 2 O 2 , according to the manufacturer's instructions.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Oncology Letters

Article Title: Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis

doi: 10.3892/ol.2022.13419

Figure Lengend Snippet: Promotive effects of ANGPTL4 on various stages of tongue cancer cell metastasis from the tongue to lungs. Black arrows indicate the stages of metastasis promoted by ANGPTL4. ANGPTL4, angiopoietin-like 4.

Article Snippet: Sections were then incubated (4°C overnight) with a rabbit polyclonal antibody to ANGPTL4 (20 μg/m1; ab196746, Abcam) and stained at room temperature for 20 min using EnVision+ solution (Dako Cytomation) and 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.006% H 2 O 2 , according to the manufacturer's instructions.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: Role of ANGPTL4 in the IMQ-induced psoriasiform dermatitis in mice. (A) Schematic illustration of the animal experiment protocol for the CON, IMQ, IMQ + A4 group ( n = 5). (B) Phenotypical presentation of mouse back skin and light microscopy examination of skin sections stained with H&E after 7 days of CON, IMQ, IMQ + A4 groups. Scale bars, 50 μm. (C) Daily assessment of epidermal erythema, scales, and thickness of the shaved backs. PASI score was calculated to assess the severity of psoriasis by adding the scores of three criteria (range from 0 to 12). (D) The skin thickness was measured on day 8. Significant differences are indicated. (E,F) Representative immunohistochemical stainings and quantification of ANGPTL4 in skin sections. Scale bars, 50 μm. A4, ANGPTL4; CON group = 62.5 mg/day Vaseline cream +25 ug/kg/day vehicle i. d. for 7 days, IMQ group = 62.5 mg/day 5% IMQ cream +25ug/kg/day vehicle i. d., IMQ + A4 group = 62.5 mg 5% IMQ cream+ 25 ug/kg/day recombinant ANGPTL4 i. d. Data were shown as mean ± standard deviation (SD); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON group.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Light Microscopy, Staining, Immunohistochemical staining, Recombinant, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: Identification of modules associated with psoriasis using the WGCNA. (A) Gene dendrogram of the top 25% most variant genes clustered based on a dissimilarity measure. In total, 12 modules were identified. (B) Module eigengene dendrogram and eigengene adjacency heatmap summarize the modules yielded in the hierarchical clustering analysis. (C) Heatmap of the module-trait correlation. (D) Volcano plot of genes detected in psoriasis. Red means up-regulated DEGs; blue means down-regulated DEGs; grey means no difference. (E) Venn diagrams of the intersection between DEGs and the hub genes in the turquoise module. (F) Box plot showing the differences in ANGPTL4 gene expression between lesional and paired non-lesional skin in the same patient. LS, psoriasis lesions skin; NL, non-lesional skin; DEG, differentially expressed gene.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Variant Assay, Expressing

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: ANGPTL4 expression is elevated in IMQ-induced psoriasiform dermatitis in mice. IMQ or control cream (vaseline) was applied daily to the dorsal skin for female BALB/c mice. (A) Phenotypical presentation of mouse back skin and light microscopy examination of skin sections stained with H&E after 7 days of control cream (upper panel) or IMQ treatment (lower panel). Scale bars, 50 μm. (B) The skin thickness was measured on day 8. Significant differences are indicated ( n = 5). (C) Daily assessment of epidermal erythema, scales, and thickeness of the shaved backs. PASI score was calculated to assess the severity of psoriasis by adding the scores of three criteria (range from 0 to 12). (D,E) Representative immunoblotting and analysis of ANGPTL4 from skin samples of control cream or IMQ treated mice on day 8 ( n = 6). (F) Relative mRNA expression of ANGPTL4 in IMQ treatment mice and control cream on day 8 ( n = 5). (G,H) Representative immunohistochemical stainings and quantification of ANGPTL4 in skin sections ( n = 5). Scale bars, 50 μm. (I,J) Representative images and quantification of ANGPTL4 fluorescence in skin sections. The nuclei were detected by DAPI (blue). Scale bar, 50 μm. All data were shown as mean ± standard deviation (SD). IMQ, imiquimod; H&E, haematoxylin and eosin; IOD, the integral optical density. * p < 0.05, ** p < 0.01 vs. CON group.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Expressing, Light Microscopy, Staining, Western Blot, Immunohistochemical staining, Fluorescence, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: ANGPTL4 expression is elevated in psoriasis patients. (A) Phenotypical presentation of psoriasis and normal skin stained with H&E. Scale bars, 250 μm. (B) The skin thickness was measured. Significant differences are indicated ( n = 5). (C,D) Representative immunohistochemical stainings and quantification of ANGPTL4 in skin sections from psoriasis and non-psoriasis patients ( n = 5). Scale bars, 250 μm. (E,F) Representative images and quantification of ANGPTL4 fluorescence in skin sections. The nuclei were detected by DAPI (blue). Scale bar, 100 μm. All data were shown as mean ± standard deviation (SD). H&E, haematoxylin and eosin; IOD, the integral optical density. ** p < 0.01 vs. CON group.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Expressing, Staining, Immunohistochemical staining, Fluorescence, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: Effect of ANGPTL4 on Human Keratinocyte Cells (A) Representative Western blots showing PCNA, Cyclin D1, Cleaved IL-1β and IL-17A in HaCaT cells after treatment with ANGPTL4 (500 ng/ml). (B) WB analysis of PCNA, Cyclin D1, Cleaved IL-1β, IL-17A expression, respectively. (C) CCK-8 assay results ( n = 10). (D–G) Relative mRNA expression of IL-1β, IL-17A, IL-6 and TNF-α normalized to a GAPDH internal control ( n = 5). Values are expressed relative to control groups. (H–K) The secretion of IL-1β, IL-17, IL-6 and TNF-α were determined by ELISA ( n = 3). Results shown are representative data of three independent experiments. All data were shown as mean ± standard deviation (SD). ** p < 0.01, *** p < 0.001 vs. CON group.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Western Blot, Expressing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: Effect of ANGPTL4 Knockdown on Human Keratinocyte Cells (A,B) Representative Western blots and quantification of ANGPTL4, PCNA, Cyclin D1, Cleaved IL-1β and IL-17A in cultured HaCaT cells under the treatment with si-RNA targeting ANGPTL4 (si-ANG-01 and si-ANG-02) or negative control siRNA (si-NC). GAPDH was used as loading control ( n = 3). (C) CCK-8 assay results ( n = 9). (D) Relative mRNA expression of IL-1β, IL-17A, IL-6 and TNF-α normalized to a GAPDH internal control ( n = 5). Results shown are representative data of three independent experiments. All data were shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON group.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Western Blot, Cell Culture, Negative Control, CCK-8 Assay, Expressing, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: Modulation of STAT3 and ERK1/2 pathways by ANGPTL4 in HaCaT cells. (A) Representative Western blots showing p-ERK1/2, ERK1/2, p-STAT3, STAT3 levels in HaCaT cells after treatment with ANGPTL4 (500 ng/ml). (B) Representative Western blots showing PCNA, Cyclin D1, Cleaved IL-1β and IL-17A in HaCaT cells after treatment with ANGPTL4 and MEK inhibitor (PD0325901). (C) Representative Western blots showing p-ERK1/2, ERK1/2, p-STAT3 and STAT3 levels in HaCaT cells under treatment with si-RNA targeting ANGPTL4 (si-ANG-01 and si-ANG-02) or negative control siRNA (si-NC). GAPDH was used as loading control ( n = 3). (D) Quantitative Western blot analysis. Results shown are representative data of three independent experiments. All data were shown as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON group.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Western Blot, Negative Control, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: ANGPTL4 Regulates Psoriasis via Modulating Hyperproliferation and Inflammation of Keratinocytes

doi: 10.3389/fphar.2022.850967

Figure Lengend Snippet: Role of ANGPTL4 in the IMQ-induced psoriasiform dermatitis in mice. (A,B) Representative Western blots and quantification showing phospho-ERK1/2, ERK1/2, phospho-STAT3, STAT3, Cleaved IL-1β and IL-17A levels in the CON, IMQ, IMQ + A4 group ( n = 3). (C) Relative mRNA expression of IL-1β, IL-17A, IL-6 and TNF-α in the CON, IMQ, IMQ + A4 group ( n = 5). Data were shown as mean ± standard deviation (SD); ** p < 0.01, *** p < 0.001 vs. CON group.

Article Snippet: After blocking in 5% bovine albumin (BSA) for 1 h, membranes were incubated with primary antibodies at against STAT3 (1:1,000, A1192, ABclonal), p-STAT3-Y705 (1:1,000, AP0705, ABclonal), ERK1/ERK2 (1:1,000, A16686, ABclonal), p-ERK1-T202/Y204 + ERK2-T185/Y187 (1:1,000, AP0472, ABclonal), PCNA (1:1,000, A0264, ABclonal), Cyclin D1 (1:1,000, 2922S, Cell Signaling Technology) Cleaved IL-1β (1:1,000, Cell Signaling Technology), IL-17A (1:1,000, A0688, ABclonal), ANGPTL4 (1:1,000, CSB-PA005044, Cusabio), ANGPTL4 (1:500, AF3485, R&D Systems), GAPDH (1:5,000, CSB-MA000071M2m, Cusabio) at 4°C overnight.

Techniques: Western Blot, Expressing, Standard Deviation