Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: PKM2 Knockdown Induces Autophagic Cell Death via AKT/mTOR Pathway in Human Prostate Cancer Cells.

doi: 10.33594/000000107

Figure Lengend Snippet: Fig. 6. Expression patterns of different signaling pathway proteins after PKM2 knockdown by siRNAs (100 nM for 72 h). (A) The effect of PKM2 knockdown on anticancer signaling pathways in DU145 cells. The cells were transfected with the indicated siRNAs, and western blotting was performed to determine the expression levels of the different signaling pathway proteins. Representative blots are shown. (B) The intensities of the bands were measured and depicted in the bar graph by the ratio of GAPDH. (C) The ratio of p-Akt/Akt, p-mTOR/mTOR, p-p70S6K/ p70S6K and p-GSK3β/ GSK3β were measured and depicted in the bar graph. One-way ANOVA was used to compare the means of different groups. Differences between the means were considered to be significant at P<0.05 by using Tukey multiple comparison tests; *P<0.05, **P<0.01, and ***P<0.001 compared with the normal control cells.

Article Snippet: Mouse monoclonal antibodies against phosphorylated P70S6K (p-P70S6K; T389) (cat. no. 9206) were purchased from Cell Signaling Technology, Inc.,.

Techniques: Expressing, Knockdown, Protein-Protein interactions, Transfection, Western Blot, Comparison, Control

Journal: Bioengineered

Article Title: Ivabradine protects rats against myocardial infarction through reinforcing autophagy via inhibiting PI3K/AKT/mTOR/p70S6K pathway

doi: 10.1080/21655979.2021.1925008

Figure Lengend Snippet: Effect of ivabradine on AKT/mTOR/p70S6K pathway of MI rats. (a) Representative WB bands of p-PI3K, PI3K, AKT and p-AKT. Quantification of p-PI3K (b) and PI3K (c), as normalized to β-actin. (d) Representative WB bands of mTOR, p-mTOR, p70S6K and p-p70S6K. Quantification of p-PI3K (e) and PI3K (f), as normalized to β-actin. n = 6 hearts/group. Data were expressed as mean ± SD; ## P < 0.01 vs Sham group; ** P < 0.01 vs MI group; & P < 0.05 vs MI+Iva group. Abbreviations: 3-MA, 3-methyladenine; Iva, ivabradine; MI, myocardial infarction; Rap, rapamycin

Article Snippet: Membranes were then blocked with 5% skimmed milk (Yili Industrial Group Co., Ltd., Inner Mongolia, China) and incubated with primary antibodies against p62 (1:1000, cat. No. 39,749, Cell Signaling Technology, Inc., Massachusetts, USA), LC3 (1:1000, cat. No. 4108, Cell Signaling Technology), Beclin-1 (1:1000, cat. No. 3495, Cell Signaling Technology), ATG-5 (1:2000, cat. No. 12,994, Cell Signaling Technology), ATG-7 (1:500, cat. No. 8558, Cell Signaling Technology), p-AKT (1:500, cat. No. 4060, Cell Signaling Technology), AKT (1:1000, cat. No. 4691, Cell Signaling Technology), p-PI3K (1:1000, cat. No. AF3242, Affinity Biosciences, Cincinnati, OH, USA), PI3K (1:1000, cat. No. 4257, Cell Signaling Technology), p-mTOR (1:500, cat. No. 5536, Cell Signaling Technology), mTOR (1:1000, cat. No. 2983, Cell Signaling Technology), p-p70S6K (1:1000, cat. No. 97,596, Cell Signaling Technology), p70S6K (1:2000, cat. No. 9202, Cell Signaling Technology) or β-actin (1:1000, cat. No. sc-47,778, Santa Cruz Biotechnology, Inc., California, USA) at 4°C overnight.

Techniques:

Journal: bioRxiv

Article Title: A novel frameshift mutation in Phosphoinositide 3-kinase regulatory subunit 1 ( PIK3R1 ) causes immunodeficiency and Amyotrophic Lateral Sclerosis (ALS)

doi: 10.1101/2025.05.23.655625

Figure Lengend Snippet: A) Immunoblot analysis of pAKT (Thr308), pAKT (Ser473), total AKT from patient’s iPSCs lysate compared to edited and healthy controls are shown. Densitometry data are pooled from 5 (patient and healthy control) and 6 (patient and edited) independent experiments showed pAKT (Thr308) and pAKT(Ser473)/Total AKT ratio are significantly elevated in patient compared to edited and healthy controls ** p<0.01,* p<0.05 respectively. B) Pooled densitometry data from 4 independent immunoblot experiments of phospho-S6 to total S6 ratio did not demonstrate any change in patient derived iPSCs compared to healthy and edited cells. C) Multi-well microelectrode array (MEA) recordings of terminally differentiated motor neurons (iMNs) shows significantly reduced mean firing rate of patient-derived iMNs compared to healthy iMNs from day 32 to the end of the experiments (Data are representative of three separate experiments; ** p<0.01). D) MEA recordings shows normalized mean firing rate of iMNs from patient’s edited iPSCs compared to patient’s iMNs from day 32 to the end of the experiments (Data are representative of three separate experiments; ** p<0.01). E) CRISPR-Cas9 PIK3R1 c.1710dup mutated iMNs exhibit reduced electrical activity compared to isogenic healthy control iMNs. (Data are representative of three separate experiments; **** p<0.0001). F-G) Immunohistochemistry staining for cleaved caspase-3 (Red) and DAPI (blue) in iMNs on Day 55 post differentiation show percentage of apoptotic cells was significantly increased in patient and PIK3R1 c.1710dup mutated iMNs compared to healthy controls (Data are representative of three separate experiments ****p < 0.0001) and Gene correction significantly decreased cleaved caspase-3 expression (Red) to total number of cells in edited iMNs compared to patient cells. (Data are representative of three separate experiments ****p<0.0001). H) CRISPR-Cas9 PIK3R1 c.1710dup mutated iMNs also showed significantly elevated cleaved caspase-3 positive cells compared to isogenic healthy control (Data are representative of three separate experiments ***p<0.001).

Article Snippet: A primary antibody in BSA was added to the membranes, which were then incubated overnight at 4 C. Primary antibodies used included: vinculin (Abcam; Cat # AB129002), Akt Rabbit Ab (Cell Signaling Technology; Cat # 9272S), phospho-Akt (Ser473) Rabbit mAb (Cell Signaling Technology; Cat #4060S), phospho-Akt (Thr308) Rabbit mAb (Cell Signaling Technology; Cat #4056S), S6 ribosomal protein Rabbit mAb (Cell Signaling Technology; Cat #2217S), phospho-S6 ribosomal protein (Ser240/244) Rabbit mAb (Cell Signaling Technology; Cat #5364S), PI3 kinase p85 alpha Rabbit mAb (Cell Signaling Technology; Cat # 4257).

Techniques: Western Blot, Control, Derivative Assay, Microelectrode Array, CRISPR, Activity Assay, Immunohistochemistry, Staining, Expressing