Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiorenal injury in a mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/CM9.0000000000002978

Figure Lengend Snippet: ILK signaling was activated after Ang-II infusion in WT and miR-26a-KO mice. (A) Representative Western blotting figure showing the levels of ILK and LIMS1 in the heart of mice. (B) Semi-quantitative statistical analysis of ILK and LIMS1 protein levels in the heart for Western blotting results ( n = 6). (C) Representative Western blotting figure showing the levels of ILK and LIMS1 in the kidney of mice. (D) Semi-quantitative statistical analysis of ILK and LIMS1 protein levels in the kidney for Western blotting results ( n = 6). (E) Representative IHC staining of ILK and LIMS1 in the heart and kidney. Scale bars: 40 μm. (F & G) Co-immunoprecipitation demonstrated an interaction between LIMS1 and ILK. Heart or kidney tissue lysates were immunoprecipitated with specific antibody against LIMS1, followed by immunoblotting with antibodies against ILK. Data are presented as mean ± SD. Ang: Angiotensin; Ctrl: Control; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IB: Immunoblotting; IHC: Immunohistochemistry; ILK: Integrin-linked kinase; IP: Immunoprecipitation; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SD: Standard deviation; WT: Wild type.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL-1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Western Blot, Immunohistochemistry, Immunoprecipitation, Control, Knock-Out, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiorenal injury in a mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/CM9.0000000000002978

Figure Lengend Snippet: Inhibition of LIMS1 significantly improved Ang-II-induced heart injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in the heart for Western blotting results ( n = 6). (B) Real-time PCR analysis of ANP, BNP and β-MHC mRNA levels ( n = 6). (C) Representative immunofluorescence staining of WGA (green) in heart. Scale bars: 40 μm. (D) Representative immunofluorescence staining of CD68 (green) in heart. Scale bars: 40 μm. (E) Representative Masson’s staining of heart. Scale bars: 20 μm. Data are presented as mean ± SD. Ang: Angiotensin; ANP: Atrial natriuretic peptide; BNP: Brain natriuretic peptide; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; WGA: Wheat germ agglutinin; WT: Wild type; α-SMA: α-smooth muscle actin; β-MHC: β-myosin heavy chain.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL-1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Inhibition, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Control, Knock-Out, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiorenal injury in a mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/CM9.0000000000002978

Figure Lengend Snippet: Inhibition of LIMS1 significantly improved Ang-II-induced kidney injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in kidney for Western blotting results ( n = 6). (B) 24-h urinary protein quantity at the 4th week of Ang-II infusion in WT and miR-26a-KO mice ( n = 6). (C) SCr levels at the 4th week of Ang-II infusion in WT and miR-26a KO mice ( n = 6). (D) Representative immunofluorescence staining of CD68 (green) in kidney. Scale bars: 40 μm. (E) Representative Masson’s staining of kidney. Scale bars: 20 μm. Data are presented as mean ± SD. Ang: Angiotensin; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; WT: Wildtype; α-SMA: α-smooth muscle actin.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL-1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Inhibition, Western Blot, Immunofluorescence, Staining, Control, Knock-Out, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiorenal injury in a mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/CM9.0000000000002978

Figure Lengend Snippet: Overexpression of miR-26a significantly improved Ang-II-induced cardiorenal injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in heart for Western blotting results ( n = 6). (B) Representative Masson’s staining of heart and kidney. Scale bars: 20 μm. (C) Representative immunofluorescence staining of CD68 (green) in heart and kidney. Scale bars: 40 μm. (D) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in kidney for Western blotting results ( n = 6). Data are presented as mean ± SD. Ang: Angiotensin; CD: Cluster of differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; α-SMA: α-smooth muscle actin.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL-1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Over Expression, Western Blot, Staining, Immunofluorescence, Control, Knock-Out, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/cm9.0000000000002978

Figure Lengend Snippet: Figure 3: ILK signaling was activated after Ang-II infusion in WT and miR-26a-KO mice. (A) Representative Western blot figure showing the levels of ILK and LIMS1 in the heart of mice. (B) Semi-quantitative statistical of ILK and LIMS1 protein levels in the heart for Western blotting results (n = 6). (C) Representative Western blot figure showing the levels of ILK and LIMS1 in the kidney of mice. (D) Semi-quantitative statistical analysis of ILK and LIMS1 protein levels in the kidney for Western blotting results (n = 6). (E) Representative IHC staining of ILK and LIMS1 in the heart and kidney. Scale bars: 40 µm. (F & G) Co-immunoprecipitation demonstrated an interaction between LIMS1 and ILK. Heart or kidney tissue lysates were immunoprecipitated with specific antibody against LIMS1, followed by immunoblotting with antibodies against ILK. Data are presented as mean ± SD. Ang: Angiotensin; Ctrl: Control; IB: Immunoblotting; IHC: Immunohistochemistry; ILK: Integrin-linked kinase; IP: Immunoprecipitation; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SD: Standard deviation; WT: Wild type.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), Fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Western Blot, Immunohistochemistry, Immunoprecipitation, Control, Knock-Out, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/cm9.0000000000002978

Figure Lengend Snippet: Figure 4: Inhibition of LIMS1 significantly improved Ang-II-induced heart injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in the heart for Western blotting results (n = 6). (B) Real-time PCR analysis of ANP, BNP and β-MHC mRNA levels (n = 6). (C) Representative immunofluorescence staining of WGA (green) in heart. Scale bars: 40 µm. (D) Representative immunofluorescence staining of CD68 (green) in heart. Scale bars: 40 µm. (E) Representative Masson’s staining of heart. Scale bars: 20 µm. Data are presented as mean ± SD. Ang: Angiotensin; ANP: Atrial natriuretic peptide; BNP: Brain natriuretic peptide; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylin- dole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; WGA: Wheat germ agglutinin; WT: Wildtype; α-SMA: α-smooth muscle actin; β-MHC: β-myosin heavy chain.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), Fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Inhibition, Western Blot, Real-time Polymerase Chain Reaction, Staining, Control, Knock-Out, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/cm9.0000000000002978

Figure Lengend Snippet: Figure 5: Inhibition of LIMS1 significantly improved Ang-II-induced kidney injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in kidney for Western blotting results (n = 6). (B) 24-h urinary protein quantity at the 4th week of Ang-II infusion in WT and miR-26a-KO mice (n = 6). (C) SCr levels at the 4th week of Ang-II infusion in WT and miR-26a KO mice (n = 6). (D) Representative immunofluorescence staining of CD68 (green) in kidney. Scale bars: 40 µm. (E) Representative Masson’s staining of kidney. Scale bars: 20 µm. Data are presented as mean ± SD. Ang: Angiotensin; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; WT: Wildtype; α-SMA: α-smooth muscle actin.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), Fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Inhibition, Western Blot, Staining, Control, Knock-Out, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

doi: 10.1097/cm9.0000000000002978

Figure Lengend Snippet: Figure 6: Overexpression of miR-26a significantly improved Ang-II-induced cardiorenal injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in heart for Western blotting results (n = 6). (B) Representative Masson’s staining of heart and kidney. Scale bars: 20 µm. (C) Representative immunofluorescence staining of CD68 (green) in heart and kidney. Scale bars: 40 µm. (D) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in kidney for Western blotting results (n = 6). Data are presented as mean ± SD. Ang: Angiotensin; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; α-SMA: α-smooth muscle actin.

Article Snippet: After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), Fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio).

Techniques: Over Expression, Western Blot, Staining, Control, Knock-Out, Standard Deviation

Journal: PLOS Pathogens

Article Title: Negative regulation of type I interferon signaling by integrin-linked kinase permits dengue virus replication

doi: 10.1371/journal.ppat.1011241

Figure Lengend Snippet: ( A-B ) The protein levels of IFN-β ( A ) and mRNA levels of MxA ( B ) in control (shLuc) or ILK knockdown (shILK) A549 cells infected without (-) or with (+) DENV at 24 hours post-infection (hpi) are shown. The MxA level in control cells without DENV infection was set as 1. Data represent mean + SD (error bars). * p < 0.05, ** p < 0.01, *** p < 0.001. ( C ) Representative western blots of phosphorylated (p-) and indicated proteins in mock- (M) and DENV-infected control or ILK knockdown A549 cells at indicated hpi are shown. The ratios of phosphorylated to total STAT1 and STAT2 relative to infected control cells at 24 hpi are shown. ( D ) Representative western blots of indicated proteins in A549 cells infected without (-) or with (+) DENV and treated with the indicated concentration of OSU-T315 for 24 hours are shown. The ratios of MxA and NS1 to β-actin as well as phosphorylated to total STAT1 relative to infected cells without OSU-T315 treatment are shown.

Article Snippet: Cell lysates were subjected to western blot analysis using primary antibodies against Akt with phosphorylation (p-) on Ser473, Akt, ILK, p701-STAT1, STAT1 (Epitomics), p690-STAT2, STAT2 (Abcam), p536-p65, p65, SOCS3 (Proteintech), p32-IκBα, IκBα, p202/204-Erk, Erk, NS1 (clone 33-D2 kindly provided by Dr. Trai-Ming Yeh), NS3 (GeneTex), NS4B (GeneTex), prM (clone 155–49), and β-actin (Abcam) and HRP-conjugated secondary antibodies.

Techniques: Control, Knockdown, Infection, Western Blot, Concentration Assay

Journal: PLOS Pathogens

Article Title: Negative regulation of type I interferon signaling by integrin-linked kinase permits dengue virus replication

doi: 10.1371/journal.ppat.1011241

Figure Lengend Snippet: ( A ) Representative western blots of the indicated proteins in mock- (M) and DENV-infected control (shLuc) or ILK knockdown (shILK) A549 cells at indicated hours post-infection (hpi) are shown. The ratios of SOCS1 or SOCS3 to β-actin relative to mock-infected control cells are shown. ( B-C ) Representative RT-PCR images of the indicated genes ( B ) and western blots of phosphorylated (p-) and indicated proteins ( C ) in control and SOCS3 knockdown (shSOCS3) A549 cells infected with (+) or without (-) DENV at indicated or 24 hpi are shown. The ratios of phosphorylated to total STAT1 and STAT2 relative to infected control cells are shown. ( D ) The viral yields in indicated A549 cells at 24 hpi are shown. Data represent mean + SD (error bars). * p < 0.05. ( E ) Representative western blots of indicated proteins in A549 cells infected with (+) or without (-) DENV and treated with the indicated concentration of OSU-T315 at 24 hpi are shown. BD, below detection.

Article Snippet: Cell lysates were subjected to western blot analysis using primary antibodies against Akt with phosphorylation (p-) on Ser473, Akt, ILK, p701-STAT1, STAT1 (Epitomics), p690-STAT2, STAT2 (Abcam), p536-p65, p65, SOCS3 (Proteintech), p32-IκBα, IκBα, p202/204-Erk, Erk, NS1 (clone 33-D2 kindly provided by Dr. Trai-Ming Yeh), NS3 (GeneTex), NS4B (GeneTex), prM (clone 155–49), and β-actin (Abcam) and HRP-conjugated secondary antibodies.

Techniques: Western Blot, Infection, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Concentration Assay