Journal: Molecular Cancer Research

Article Title: AKT1 E17K Inhibits Cancer Cell Migration by Abrogating β-Catenin Signaling

doi: 10.1158/1541-7786.mcr-20-0623

Figure Lengend Snippet: Figure 4. AKT2, but not AKT1, increases the transcriptional activity of b-catenin on the ZEB1 promoter, and decreases E-cadherin expression. A, p53ko/E17K cells were infected with a CRISPR/Cas9 lentivirus targeting AKT1 and p53ko cells were infected with a CRISPR/Cas9 lentivirus targeting AKT2. Cells lysates were then subjected to ChIP using a b-catenin antibody. Top: Western blot of whole cell lysates from the indicated p53ko/E17K cells and p53ko cells before and after AKT1 knockdown (AKT1KD) or AKT2 knockdown (AKT2KD) as specified. Bottom: Relative fold of ChIP pull-down of the ZEB1 promoter (b-catenin fold enrichment) was quantified by real-time qPCR, normalized by both input DNA and IgG values. Error bars, mean SEM (n ¼ 3). P values were determined by one-way ANOVA followed by Tukey post hoc test. , P < 0.0001. B, Western blots of lysates from MCF-10A p53ko/E17K or MCF7 PIK3CA WT cells expressing HA-tagged Myr-AKT2 and FLAG-tagged Myr-AKT1 alone and in combination. C, Western blot of lysates from p53ko cells: Left: infected with PTEN CRISPR/Cas9 lentivirus; Right: infected with PIK3CA E545K lentivirus. Cell lysate of parental MCF-10A cells was used as an E-cadherin–positive control.

Article Snippet: PIK3CA E545K–mutant cDNA (Cat#RC400348), purchased from OriGene, was inserted into the AsiS I and Mlu I sites of plasmid Lenti-C-Myc-DDK-IRES-Neo (Cat#PS100081, OriGene), to generate PIK3CA-E545K lentiviral expression vector.

Techniques: Activity Assay, Expressing, Infection, CRISPR, Western Blot, Knockdown, Positive Control

Journal: The Journal of international medical research

Article Title: The therapeutic potential of irisin in alleviating acute lung injury via inflammation and ferroptosis modulation.

doi: 10.1177/03000605251340338

Figure Lengend Snippet: Figure 6. Western blotting analysis on the effects of irisin on the expression level of ferroptosis-associated proteins. (a) Representative western blotting images for the expression of ACSL4, COX-2, GPX4, p-AMPK, and t-AMPK in lung tissues. GAPDH was selected as the loading control protein. (b) Quantification analysis of the related bands of ACSL4, COX-2, GPX4, p-AMPK, and t-AMPK in lung tissues. Lung tissues were harvested on day 3 post-CLP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. n ¼ 6 per group. Data are presented as means SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Subsequently, the membranes were incubated with rabbit anti-AMPK (MCE, Cat# HY-P80541), anti-p-AMPK (MCE, HYP80452), GPX4 (MCE, HY-P80450), ACSL4 (Santa Cruz Biotechnology, Dallas, Texas, USA; Cat# sc-365230), COX-2 (Abcam, Cambridge, United Kingdom; Cat# ab283574), and GAPDH (MCE, HY-P80137) antibodies at 4 C overnight.

Techniques: Western Blot, Expressing, Control

Journal: Oncology Reports

Article Title: SNX10 regulates the proliferation, apoptosis and cell cycle of acute B lymphoblastic leukemia cells via the PI3K/Akt signaling pathway

doi: 10.3892/or.2025.8911

Figure Lengend Snippet: SNX10 promotes B-ALL cellular behaviors via the PI3K/Akt signaling pathway. (A) Venn diagram showing the 211 significantly differentially expressed proteins identified between SNX10-OE Rs4;11 and EV cells. (B) Volcano plot of the differentially expressed proteins showed 76 downregulated and 135 upregulated proteins. (C) KEGG pathway enrichment analysis of the differentially expressed proteins. SNX10, sorting nexin 10; B-ALL, B-cell acute lymphoblastic leukemia; OE, overexpression; EV, empty vector; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Idelalisib (IDEL, TargetMoi, Cas: 870281-82-6) is a small molecule inhibitor that targets the PI3K catalytic subunit p110δ.

Techniques: Analysis, Over Expression, Plasmid Preparation, Significance Assay

Journal: Oncology Reports

Article Title: SNX10 regulates the proliferation, apoptosis and cell cycle of acute B lymphoblastic leukemia cells via the PI3K/Akt signaling pathway

doi: 10.3892/or.2025.8911

Figure Lengend Snippet: SNX10 influences the activity of the PI3K/Akt signaling pathway in B-ALL cells. The protein expression levels of PI3K, p-PI3K, Akt and p-Akt proteins in SNX10-OE and knockdown (A) Rs4;11 and (B) Nalm-6 cells were detected. After treating SNX10-OE B-ALL cells with 10 µM IDEL (a PI3K inhibitor) for 24 h, the protein expression levels of PI3K, p-PI3K, Akt and p-Akt proteins in SNX10-OE and knockdown (C) Rs4;11 and (D) Nalm-6 cells were detected. Data are presented as the mean ± SD of three independent repeats and compared using a one-way or two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. EV group or shNC group. SNX10, sorting nexin 10; B-ALL, B-cell acute lymphoblastic leukemia; OE, overexpression; EV, empty vector; shRNA, short hairpin RNA; NC, negative control; IDEL, Idelalisib; p-, phosphorylated.

Article Snippet: Idelalisib (IDEL, TargetMoi, Cas: 870281-82-6) is a small molecule inhibitor that targets the PI3K catalytic subunit p110δ.

Techniques: Activity Assay, Expressing, Knockdown, Over Expression, Plasmid Preparation, shRNA, Negative Control, Western Blot

Journal: Oncology Reports

Article Title: SNX10 regulates the proliferation, apoptosis and cell cycle of acute B lymphoblastic leukemia cells via the PI3K/Akt signaling pathway

doi: 10.3892/or.2025.8911

Figure Lengend Snippet: SNX10 affects the activity of B-ALL cells through the PI3K/Akt signaling pathway. After treating (A) Rs4;11 cells and (B) Nalm-6 cells with 10 µM IDEL (a PI3K inhibitor) for 24 h, cell proliferation was analyzed using a Cell Counting Kit-8 assay. Apoptosis of (C) Rs4;11 cells and (D) Nalm-6 cells (annexin V/7-AAD staining) was analyzed by flow cytometry. Cell cycle distribution in (E) Rs4;11 cells and (F) Nalm-6 cells was analyzed by flow cytometry. Data are presented as the mean ± SD of three independent repeats and compared using a one-way or two-way ANOVA. ***P<0.001 and ****P<0.0001 vs. EV group. SNX10, sorting nexin 10; B-ALL, B-cell acute lymphoblastic leukemia; OE, overexpression; EV, empty vector; IDEL, Idelalisib; ns, not significant.

Article Snippet: Idelalisib (IDEL, TargetMoi, Cas: 870281-82-6) is a small molecule inhibitor that targets the PI3K catalytic subunit p110δ.

Techniques: Activity Assay, Cell Counting, Staining, Flow Cytometry, Over Expression, Plasmid Preparation, FACS

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: A,C: Effect of 46 nM ASPD (A) or 100 nM ouabain (C) on carbachol-induced relaxation of ex-vivo rat aortic rings (n = 3 for mASD3-preincubated ASPD-treated group, and n = 5 for other groups). B: Representative images of immunohistochemical multiple staining of NAKα3 and vWF in the endothelial layer of rat aortic rings. The arrowheads indicate NAKα3 on the apical surface of endothelium. Scale bars: 1 μm. In (A,C), data are presented as means ± S.E. ** P < 0.01 (ANOVA with Scheffé’s method (A) and Welch’s t -test (C)).

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Ex Vivo, Immunohistochemical staining, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: A: Representative 2D images of immunocytochemical staining of NAKα3 and nuclei (DAPI) on human primary brain microvessel endothelial cells (left) and vertical section image prepared from z-stack 3D image (right). Scale bars: 5 μm. B: Western blotting for NAKα3 in the endothelial cells (left) and agarose electrophoresis of RT-PCR products for ATP1A3 mRNA (right). C: Binding ratio of ASPD signal to total NAKα3 signal (the representative images in upper panels and the quantification in bottom, n = 5). Scale bars: 5 μm. D: High-power representative 2D images of multiple immunocytochemical staining of ASPD (ASPD-specific, mASD3 antibody), NAKα3, and nuclei (DAPI) on 30 nM ASPD-treated endothelial cells (left). The vertical section image prepared from z-stack 3D image (right upper) and its line-scan analysis of fluorescence intensities of NAKα3 (green line) and ASPD (red line) (right bottom). Scale bars: 5 μm for solid line and 1 μm for hatched line. E: Western blotting for NAKα3 in the siRNA-transfected endothelial cells (left, n = 3) and quantification of number of punctate NAKα3 signal on the endothelial cells (right, n = 5). Scale bars: 5 μm. F: Effect of siRNA transfection on the binding ratio of ASPD signal to total NAKα3 signal on the endothelial cells (n = 5). The transfection of ATP1A3 siRNA decreased the ASPD binding ratio to 37 ± 12%, 11 ± 2%, 42 ± 2%, and 66.2 ± 2% in the 3 nM, 10 nM, 30 nM, and 100 nM ASPD-treated groups, respectively. In (C,E,F), data are presented as means ± S.E. * P < 0.05 (ANOVA with Scheffé’s method).

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Staining, Western Blot, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Analysis, Fluorescence, Transfection, Positive Control, SDS Page, Immunofluorescence, Polymerase Chain Reaction, Knockdown

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: High-power representative 2D images of immunocytochemical multiple staining of ASPD (ASPD-specific, mASD3 antibody), NAKα3, and nuclei (DAPI) on 30 nM ASPD-treated human primary brain microvessel endothelial cells with or without siRNA transfection (Upper, middle, and bottom panels show non-treated, mock siRNA-treated, and ATP1A3 siRNA-treated cells, respectively). Scale bars: 5 μm.

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Staining, Transfection, Immunofluorescence, Knockdown

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: ASPD bind to cell-surface NAKα3 on cerebral microvessel endothelial cells, promote mitochondrial ROS production, activate PKC, increase eNOS-Thr 495 phosphorylation, and attenuate NO release, resulting in suppression of blood microvessel relaxation response.

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Phospho-proteomics

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a Percentage of PI3K members ( PTEN , PIK3CA , PIK3CB, and PIK3R1 ) alterations in metastatic castration-resistant prostate cancer cohort (SU2C Cell 2015). b Oncoprint showing PI3K member alterations in all PI3K-altered tumors from the same cohort ( n = 76). c Venn diagram showing the percentage of overlaps among alterations in PIK3CA , PIK3CB , PIK3R1 , and PTEN . d Oncoprint showing PIK3CA , PIK3R1 , and PTEN alterations in a collection of PDOs. e Western blot showing pAKT level and PTEN status in a collection of PDOs. f Western blot showing AR and pAKT levels in PDOs treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. Quantification of pAKT473 and pAKT308 normalized to β-actin. g Western blot showing pAKT levels in isogenic PDO pairs MSK-PCa15 ( PTEN -intact, PTEN -KO) and MSK-PCa16 ( PTEN -intact, PTEN -KO) treated with BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. h Western blot showing pAKT levels in isogenic LNCaP-EV and LNCaP- PTEN pairs treated with doxycycline (500 ng/mL) for 8 h for PTEN induction, followed by either BYL719 (1 µM), AZD8186 (250 nM), or Veh for 4 h. All assays were performed with three biological replicates. Source data are provided as Source data file.

Article Snippet: pLenti-Lentiviral vector (pLenti-C-Myc-DDK-P2A-Neo) (Origene, PS100081) was used to express PIK3CA wild-type (Origene, RC213112), PIK3CA mutant (E545K) (Origene, RC400348), and PIK3CB wild-type (Origene, RC215433).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Defining the therapeutic selective dependencies for distinct subtypes of PI3K pathway-altered prostate cancers

doi: 10.1038/s41467-021-25341-9

Figure Lengend Snippet: a Western blot showing levels of AKT signaling in MSK-PCa12 organoids treated with ipatasertib (500 nM) or BYL719 (1 µM) + AZD8186 (250 nM) for 4 h. b CellTiter-Glo assay showing cell viability of MSK-PCa12 organoids treated with BYL719 (1 µM) + AZD8186 (250 nM), enzalutamide (10 µM), ipatasertib (500 nM), enzalutamide + ipatasertib, or Veh, Day 7. c Growth of MSK-PCa12 tumors in SCID mice treated with enzalutamide (10 mg/kg), ipatasertib (50 mg/kg), BYL719 (25 mg/kg) + AZD8186 (75 mg/kg), ipatasertib + enzalutamide, BYL719 + AZD8186 + enzalutamide or Veh. Some of mice were castrated when tumor reached ~200 mm 3 ( n = 5 mice per group) *** p -value = 0.0011. d Growth of CWR22Pc tumors in SCID mice treated with BYL719 (25 mg/kg), ipatasertib (50 mg/kg) or Veh. Some of mice were castrated when tumor reaches ~200 mm 3 ( n = 5 mice per group) Castration + BYL719 vs. Castration + Ipatasertinib, week 2: p -value = 0.045, week 3: p -value = 0.0086, week 4: p -value = 0.012, week 5: p -value = 0.046. e Western blot showing PI3K/AKT/MAPK signaling in CWR22Pc tumors from ( d ). f – h Heat map showing drug sensitivity of ipatasertib (500 nM) or BYL719 (1 µM) + AZD8186 (250 nM) on day 7, in cells characterized as PTEN wild type with PIK3CA mutants or RTK hyperactive ( f ), PIK3CA mutants with PTEN -loss ( g ), and PIK3CA wild type with PTEN -loss ( h ). Genetically manipulated lines are isogenic pairs. i Mechanism model of PI3K pathway alterations and response to clinically available therapeutic agents. All assays were performed with three biological replicates. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, n.s: not significant, b : one-way ANOVA compared to Veh group, error bar represents mean values ± SD. c , d : multiple t -test, two-sided error bar represents mean values ± SEM.

Article Snippet: pLenti-Lentiviral vector (pLenti-C-Myc-DDK-P2A-Neo) (Origene, PS100081) was used to express PIK3CA wild-type (Origene, RC213112), PIK3CA mutant (E545K) (Origene, RC400348), and PIK3CB wild-type (Origene, RC215433).

Techniques: Western Blot, Glo Assay