Journal: Journal of Cellular and Molecular Medicine

Article Title: The preventive and therapeutic effects of AAV1‐KLF4‐shRNA in cigarette smoke‐induced pulmonary hypertension

doi: 10.1111/jcmm.16194

Figure Lengend Snippet: (A) Expression of KLF4 in the pulmonary vessels of rats in each group. Scale bar: 30 μm (B) Expression of PCNA in the pulmonary vascular smooth muscle of rats in each group. Scale bar: 25 μm (C) Representative bands of protein expression for KLF4 and PCNA in rat pulmonary vessels, and comparison of relative protein levels (normalized to GAPDH level). AU, arbitrary units. (D) Expression of P‐AKT in the pulmonary vessels of rats in each group. Scale bar: 20 μm. Sham (n = 8), saline (n = 6), AAV1‐control vector group (n = 6) and AAV1‐KLF4‐shRNA group (n = 11) at the end of the prevention experiment. Multiple comparisons were performed by one‐way ANOVA with SNK‐q. ** P < .01, *** P < .001

Article Snippet: Furthermore, the expression of KLF4 and P‐AKT was assessed by immunostaining with antibodies against KLF4 (ProteinTech, Wuhan, China) and P‐AKT (Gene Tex, San Antonio, USA).

Techniques: Expressing, Plasmid Preparation, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: The preventive and therapeutic effects of AAV1‐KLF4‐shRNA in cigarette smoke‐induced pulmonary hypertension

doi: 10.1111/jcmm.16194

Figure Lengend Snippet: (A) Expression of KLF4 in the pulmonary vessels of rats in each group. Scale bar: 30 μm (B) Expression of PCNA in the pulmonary vascular smooth muscle of rats in each group. Scale bar: 25 μm (C) Representative bands of protein expression for KLF4 and PCNA in rat pulmonary vessels, and comparison of relative protein levels (normalized to GAPDH level). AU, arbitrary units. (D) Expression of P‐AKT in the pulmonary vessels of rats in each group. Scale bar: 20 μm. Sham (n = 6), saline (n = 7), AAV1‐control vector group (n = 7) and AAV1‐KLF4‐shRNA group (n = 8) at the end of the therapeutic experiment. Multiple comparisons were performed by one‐way ANOVA with SNK‐q. * P < .05, ** P < .01, *** P < .001

Article Snippet: Furthermore, the expression of KLF4 and P‐AKT was assessed by immunostaining with antibodies against KLF4 (ProteinTech, Wuhan, China) and P‐AKT (Gene Tex, San Antonio, USA).

Techniques: Expressing, Plasmid Preparation, shRNA

Journal: Heliyon

Article Title: The effect of PINK1/Parkin pathway on glucose homeostasis imbalance induced by tacrolimus in mouse livers

doi: 10.1016/j.heliyon.2023.e15536

Figure Lengend Snippet: Hepatic insulin resistance was induced by TAC. (A) mRNA expression. (B) Levels of mouse proteins associated with insulin resistance in liver tissue. n = 8, * P < 0.05, ** P < 0.01, in comparison with the control group. Groups: Control, saline solution; TAC low-dose (0.5 mg/kg/d); TAC medium-dose (1.0 mg/kg/d); TAC high-dose (5.0 mg/kg/d). TAC, tacrolimus; INSR, insulin receptor; IRS2, insulin receptor substrate 2; GLUT2, glucose transporter type 2; AKT2, protein kinase B beta; pAKT2, phosphorylated AKT2.

Article Snippet: In this study, the antibodies used were: anti-PINK1 (catalog no. 23274-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-Parkin (catalog no. 14060-1-AP, 1:1000, Proteintech), anti-protein kinase B beta (AKT2) (catalog no. 17609-1-AP, 1:1000, Proteintech), anti-phospho-AKT2 (pAKT2) (Ser473) (catalog no. 28731-1-AP, 1:1000, Proteintech), anti-insulin receptor (INSR) (catalog no. 20433-1-AP, 1:1000, Proteintech), anti-glucose transporter type 2 (GLUT2) (catalog no. 20436-1-AP, 1:1000, Proteintech), anti-insulin receptor substrate 2 (IRS2) (catalog no. 20702-1-1AP, 1:1000, Proteintech), and anti-β-actin (catalog no. 66009-1-Ig, 1:1000, Proteintech).

Techniques: Expressing, Comparison, Control, Saline

Journal: Heliyon

Article Title: The effect of PINK1/Parkin pathway on glucose homeostasis imbalance induced by tacrolimus in mouse livers

doi: 10.1016/j.heliyon.2023.e15536

Figure Lengend Snippet: PINK1 and its downstream molecules are targeted by TAC. (A) Levels of hepatic insulin resistance-related proteins, in the presence or absence of PINK1, post-TAC administration. (B) Schematic of the hypothetical mechanism by which TAC activates PINK1/Parkin signaling, leading to downregulated expression of downstream molecules. TAC, tacrolimus; PINK1, PTEN-induced novel kinase 1; INSR, insulin receptor; IRS2, insulin receptor substrate 2; GLUT2, glucose transporter type 2; AKT2, protein kinase B beta; pAKT2, phosphorylated AKT2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: In this study, the antibodies used were: anti-PINK1 (catalog no. 23274-1-AP, 1:1000, Proteintech, Rosemont, IL, USA), anti-Parkin (catalog no. 14060-1-AP, 1:1000, Proteintech), anti-protein kinase B beta (AKT2) (catalog no. 17609-1-AP, 1:1000, Proteintech), anti-phospho-AKT2 (pAKT2) (Ser473) (catalog no. 28731-1-AP, 1:1000, Proteintech), anti-insulin receptor (INSR) (catalog no. 20433-1-AP, 1:1000, Proteintech), anti-glucose transporter type 2 (GLUT2) (catalog no. 20436-1-AP, 1:1000, Proteintech), anti-insulin receptor substrate 2 (IRS2) (catalog no. 20702-1-1AP, 1:1000, Proteintech), and anti-β-actin (catalog no. 66009-1-Ig, 1:1000, Proteintech).

Techniques: Expressing

Journal: Respiratory Research

Article Title: AKT2 drives cancer progression and is negatively modulated by miR-124 in human lung adenocarcinoma

doi: 10.1186/s12931-020-01491-0

Figure Lengend Snippet: AKT2 is overexpressed in human NSCLC tissues. ( a and c ) Relative quantification of AKT2 expression was analyzed by qRT-PCR assay in 45 NSCLC tissues (T) and adjacent noncancerous tissues (N). ACTB mRNA levels were used as internal control for normalization of AKT2 mRNA expression. b Western blot analysis was performed to detect the AKT2 protein expression in 8 selected NSCLC tissues and adjacent tissues (left) and the quantification of relative AKT2 protein levels were shown in histogram (right). β-actin was used as internal control. d - f The public datasets from GEO (GSE 10072, GSE 31210 and GSE 32863) were used to verify AKT2 mRNA levels in NSCLC. g The public data from TIMER database was used to detect AKT2 mRNA expression in both LUAD tissues and LUSC tissues. h Tissue and normal samples from randomly selected patients with NSCLC were either stained with Hematoxylin and eosin (HE) (upper, n = 2 per group) or immunohistochemically (IHC) stained with an AKT2 antibody (bottom, n = 2 per group). Scale bar, 200 um. T: NSCLC tumor tissues N: Adjacent normal tissues. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: These sections were then incubated with an anti-AKT2 polyclonal antibody (diluted 1:200, Proteintech, 17,609–1-AP) and an anti-KI67 antibody (diluted 1:400, Cell Signaling Technology, 9449) at 4 °C overnight and visualized using diaminobenzidine as the chromogenic substrate (DAB kit, ZSGB-Biotechnology Co., Ltd., Beijing, China).

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Control, Western Blot, Staining

Journal: Respiratory Research

Article Title: AKT2 drives cancer progression and is negatively modulated by miR-124 in human lung adenocarcinoma

doi: 10.1186/s12931-020-01491-0

Figure Lengend Snippet: Up-regulated AKT2 expression is associated with poor prognosis in LUAD patients. a and b Kaplan-Meier plots from Kaplan-Meier plotter shows the correlation between AKT2 expression and overall survival ( a ) or progression-free survival in patients with NSCLC ( b ). c - f Kaplan-Meier plotter data set shows the correlation between AKT2 expression and the overall survival or progression-free survival in LUAD ( c , d ) and LUSC ( e , f ) patients. g and h Public data from TIMER database shows the correlation between AKT2 expression and overall survival in LUAD ( g ) and LUSC ( h ) patients. OS, overall survival; PFS, progression-free survival. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: These sections were then incubated with an anti-AKT2 polyclonal antibody (diluted 1:200, Proteintech, 17,609–1-AP) and an anti-KI67 antibody (diluted 1:400, Cell Signaling Technology, 9449) at 4 °C overnight and visualized using diaminobenzidine as the chromogenic substrate (DAB kit, ZSGB-Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing

Journal: Respiratory Research

Article Title: AKT2 drives cancer progression and is negatively modulated by miR-124 in human lung adenocarcinoma

doi: 10.1186/s12931-020-01491-0

Figure Lengend Snippet: Knockdown of AKT2 significantly prevents cell proliferation in LUAD cell lines. a and b AKT2 levels were detected by western blot ( a ) and qRT-PCR ( b ) in multiple NSCLC cell lines. c and d A549 and H1299 cells were transfected with control siRNA or siRNA against AKT2, the effective knock down of AKT2 expression was validated by western blot ( c ) and qRT-PCR ( d ). e and f CCK-8 assay was performed to detect the cell growth in AKT2 silenced A549 ( e ) and H1299 ( f ) cells. g Colony formation abilities were detected in AKT2 silenced A549 and H1299 cells. Representative images of colonies for cell proliferation were shown (left). The colony numbers in AKT2 silenced cells were normalized to negative control (right). h An EdU staining assay was performed to determine the proliferation ability of AKT2 silenced A549 and H1299 cells (left). EdU-positive ratio were shown (right). Scale bar, 2 mm. Each experiment was performed in triplicate independently. Student’s t-test was used for statistical analysis and data are presented as the mean ± SD.* P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: These sections were then incubated with an anti-AKT2 polyclonal antibody (diluted 1:200, Proteintech, 17,609–1-AP) and an anti-KI67 antibody (diluted 1:400, Cell Signaling Technology, 9449) at 4 °C overnight and visualized using diaminobenzidine as the chromogenic substrate (DAB kit, ZSGB-Biotechnology Co., Ltd., Beijing, China).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Transfection, Control, Expressing, CCK-8 Assay, Negative Control, Staining

Journal: Respiratory Research

Article Title: AKT2 drives cancer progression and is negatively modulated by miR-124 in human lung adenocarcinoma

doi: 10.1186/s12931-020-01491-0

Figure Lengend Snippet: Silencing AKT2 attenuates the cell cycle progression of LUAD cells without affecting apoptosis. a Flow cytometry cell cycle analysis was performed in A549 and H1299 cells transfected with Si-NC, Si-AKT2–1 and Si-AKT2–3. The distribution (%) of G0/G1, S and G2/M phases are shown in the histograms (bottom). b Flow cytometry apoptosis assay was performed in A549 and H1299 cells transfected with Si-NC, Si-AKT2–1 and Si-AKT2–3. 48 h post-transfection, cells were harvested and stained with Annexin V/FITC and propidium iodide (PI). The percentage of apoptotic cells are shown in the right panel. The date values represent mean ± SD of three measurements independently. NS: no significance. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: These sections were then incubated with an anti-AKT2 polyclonal antibody (diluted 1:200, Proteintech, 17,609–1-AP) and an anti-KI67 antibody (diluted 1:400, Cell Signaling Technology, 9449) at 4 °C overnight and visualized using diaminobenzidine as the chromogenic substrate (DAB kit, ZSGB-Biotechnology Co., Ltd., Beijing, China).

Techniques: Flow Cytometry, Cell Cycle Assay, Transfection, Apoptosis Assay, Staining

Journal: Respiratory Research

Article Title: AKT2 drives cancer progression and is negatively modulated by miR-124 in human lung adenocarcinoma

doi: 10.1186/s12931-020-01491-0

Figure Lengend Snippet: Depletion of AKT2 inhibits cell migration and invasion in LUAD cells. a AKT2-silenced A549 and H1299 cells were allowed to migrate through an 8-μM pore in Transwells. The migratory cells were stained and counted in at least three microscopic fields. Cells were then treated as above and allowed to invade through the matrigel-coated membrane in the transwell inserts. Invaded cells were stained and counted under a microscope. Representative images (left) and the migratory cell numbers (right) were shown. Scale bar, 2 mm. b The mRNA expression of CDH1, CDH2, VIM, SNAI1, SNAI2, MMP7, MMP9, ZEB1 and ZEB2 were detected by qRT-PCR in AKT2 silenced A549 (upper) and H1299 (bottom) cells. c The expression of N-cadherin, Vimentin, Slug, MMP2, MMP7, MMP9, AKT, p-AKT, Erk and p-Erk protein levels were examined by western blot in AKT2 silenced A549 (left) and H1299 (right) cells. Each experiment was performed in triplicate independently and values are expressed as mean ± SD. * P < 0.05 ** P < 0.01 *** P < 0.001

Article Snippet: These sections were then incubated with an anti-AKT2 polyclonal antibody (diluted 1:200, Proteintech, 17,609–1-AP) and an anti-KI67 antibody (diluted 1:400, Cell Signaling Technology, 9449) at 4 °C overnight and visualized using diaminobenzidine as the chromogenic substrate (DAB kit, ZSGB-Biotechnology Co., Ltd., Beijing, China).

Techniques: Migration, Staining, Membrane, Microscopy, Expressing, Quantitative RT-PCR, Western Blot

Journal: Respiratory Research

Article Title: AKT2 drives cancer progression and is negatively modulated by miR-124 in human lung adenocarcinoma

doi: 10.1186/s12931-020-01491-0

Figure Lengend Snippet: MiR-124 directly binds to AKT2 3’UTR region and thus inhibits LUAD cell proliferation, migration and invasion. a Authoritative bioinformatic databases (miRDB, miRWalk, miRTarBase and TargetScan) were used to predict the putative miRNAs targeting AKT2. b After transfection with miR-124 mimics and miR-NC (left) or miR-124 inhibitor or inhibitor-NC (right) in A549 and H1299 cell lines, the mRNA and protein levels of AKT2 were analyzed by western blot and qRT-PCR assays. c Computational algorithms predict that AKT2 3’UTR region harbors a putative miR-124 binding site (upper). Reporter vector containing wild type AKT2 3’UTR fragment was constructed, and different mutation was then introduced into the potential miR-124 targeting site to obtain mutant type 3′UTR. The generated luciferase reporter plasmids were co-transfected with negative control (miR-NC) or miR-124 mimics into A549 and H1299 cells. The relative firefly luciferase activity was determined and normalized to the Renilla luciferase activity (bottom). d qRT-PCR analysis of miR-124 expression in 45 paired NSCLC tissues and adjacent normal tissues. e Kaplan-meier analysis was used to detect the correlation between miR-124 and AKT2 expression in 45 paired NSCLC tissues and adjacent normal tissues. miR-124 and AKT2 mRNA levels are normalized against U6 and β-actin, respectively. X and y axes represent the log 10 transformed T/N expression ratios of miR-124 and AKT2 mRNA, respectively. f and g CCK8 ( f ) and colony formation ( g ) assays were used to determine the proliferation abilities of miR-124 mimics or negative control (Si-NC) transfected A549 and H1299 cells. h and i miR-124 overexpressed A549 and H1299 cells were allowed to migrate through an 8-μm pore or invade through matrigel-coated membrane in transwells. Migratory and invasive cells were stained and counted in at least three light microscopic fields. Representative images ( h ) and migratory or invasive cell numbers ( i ) were shown. Scale bar, 2 mm. j and k Flow cytometry cell cycle analysis of A549 and H1299 cell lines after miR-124 overexpression (left). The distribution (%) of G0/G1, S and G2/M phases are shown in the histograms (right). l and m Indicated EMT-related mRNAs ( l ) and proteins ( m ) were analyzed by qRT-PCR and western blot in miR-124 overexpressed A549 and H1299 cells. Each analysis was performed in triplicate and Values are represented as means ± SD.* P < 0.05 ** P < 0.01 *** P < 0.001

Article Snippet: These sections were then incubated with an anti-AKT2 polyclonal antibody (diluted 1:200, Proteintech, 17,609–1-AP) and an anti-KI67 antibody (diluted 1:400, Cell Signaling Technology, 9449) at 4 °C overnight and visualized using diaminobenzidine as the chromogenic substrate (DAB kit, ZSGB-Biotechnology Co., Ltd., Beijing, China).

Techniques: Migration, Transfection, Western Blot, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Construct, Mutagenesis, Generated, Luciferase, Negative Control, Activity Assay, Expressing, Transformation Assay, Membrane, Staining, Flow Cytometry, Cell Cycle Assay, Over Expression

Journal: Respiratory Research

Article Title: AKT2 drives cancer progression and is negatively modulated by miR-124 in human lung adenocarcinoma

doi: 10.1186/s12931-020-01491-0

Figure Lengend Snippet: MiR-124 inhibits tumor growth by targeting AKT2 in vivo. a and b 2 × 10 6 A549 cells were injected into nude mice ( n = 2 mice per group). After tumor formation, miR-124 agomir and miR-NC agomir were injected into each tumor. Thirty-six days later, mice were euthanasia and tumors were surgically resected. Representative images of mice ( a ) and tumors ( b ) are shown. c The xenograft tumor volumes were measured every 2–3 days for total 36 days. Graph of tumor growth curves at the experimental endpoint. d Quantification of tumor weights from control and miR-124 agomir group. e and f qRT-PCR analysis of miR-124 and AKT2 mRNA expression in each excised tumor. U6 and ACTB were used as internal controls, respectively. g Representative images of H&E staining and IHC staining show tumor cells and the expression of AKT2 and KI67 in excised tumors from control or miR-124 agomir group. Scale bar, 200 um. * P < 0.05 ** P < 0.01 *** P < 0.001

Article Snippet: These sections were then incubated with an anti-AKT2 polyclonal antibody (diluted 1:200, Proteintech, 17,609–1-AP) and an anti-KI67 antibody (diluted 1:400, Cell Signaling Technology, 9449) at 4 °C overnight and visualized using diaminobenzidine as the chromogenic substrate (DAB kit, ZSGB-Biotechnology Co., Ltd., Beijing, China).

Techniques: In Vivo, Injection, Control, Quantitative RT-PCR, Expressing, Staining, Immunohistochemistry

Journal: Theranostics

Article Title: FKBP4 connects mTORC2 and PI3K to activate the PDK1/Akt-dependent cell proliferation signaling in breast cancer

doi: 10.7150/thno.35561

Figure Lengend Snippet: FKBP4 affects phosphorylation of Akt at Ser473 and Thr308, and activation of the PI3K/Akt signaling pathway. (A) MDA-MB-231 cells, transfected with FKBP4 or negative control siRNA, were serum deprived overnight and stimulated with 10% FCS for 1 h, or after serum deprivation, cells were deprived of amino acids with PBS for 2 h, followed by stimulation with 100ng/mL EGF, 100 nM insulin or 2X amino acids for 1 h. Cell lysates were analyzed by immunoblotting using the antibodies indicated. (B) MDA-MB-436, MCF7 and T47D cells were treated as in A. The phosphorylation of Akt in cell lysates were detected by Western blot. (C) Quantification of Western blot presented in A was performed for pS473-Akt, pT308-Akt and pS9-GSK-3β. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The antibodies were purchased from the following suppliers: FKBP4 (ProteinTech); anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pT286 Cyclin D1, anti-Cyclin D1, anti-E2F-1, anti-pT37/46 4E-BP1, anti-pS9 GSK-3β, anti-GSK-3β, and anti-mTOR (Cell Signaling Technology); anti-GAPDH and anti-Actin (Santa Cruz Biotechnology); anti-Tubulin and anti-PIK3R2 (Sigma-Aldrich).

Techniques: Activation Assay, Transfection, Negative Control, Western Blot