Journal: Global Advances in Integrative Medicine and Health

Article Title: A Prospective, Interventional, Randomized, Double-Blinded, Placebo-Controlled, Monocentric Clinical Study to Evaluate the Efficacy and Safety of Alkalihalobacillus clausii 088AE in Resolution of Acute Allergic Rhinitis Symptoms

doi: 10.1177/27536130251321821

Figure Lengend Snippet: Changes in Immunological Parameters of Patients With Allergic Rhinitis After Intervention With the Alkalihalobacillus clausii 088AE and Placebo at the Start of Treatment (SOT) to End of Treatment (EOT). Results Expressed as mean ± SE.

Article Snippet: Correspondingly, the changes in cytokines such as interleukin (IL)–IL4, IL10, IL17, and IL22, and interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and -beta (TNF-β), and Forkhead box P3 (FOXP3) [Determined by ELISA Method on an ELISA plate reader (Spectramax I3X plate reader, Molecular Devices).

Techniques:

Journal: bioRxiv

Article Title: A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation

doi: 10.1101/2021.09.17.460854

Figure Lengend Snippet: a , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

Article Snippet: The PI-PolyPIPosomes for MST were purchased from Echelon Biosciences, including PI PolyPIPosomes (#Y-P000), PI4,5P 2 PolyPIPosomes (#Y-P045), and PI3,4,5P 3 PolyPIPosomes (#Y-P039).

Techniques: Mutagenesis, Transfection, Expressing, Marker

Journal: bioRxiv

Article Title: A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation

doi: 10.1101/2021.09.17.460854

Figure Lengend Snippet: a-b , Triple fluorescent IP-WB detects stress-induced Akt association with the p53-PI3,4,5P 3 complex. MDA-MB-231 cells were treated with 30 µM cisplatin or control vehicle for 24 before being processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting Akt, PI3,4,5P 3, and p53 simultaneously. The p53 associated PI3,4,5P 3 and Akt were quantified ( b ). N=3. c , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against Akt. The Akt-associated p53 and the input of whole cell lysates were analyzed by WB. Representative images of three independent experiments are shown. d-e , IF staining of p53 and pAkt S473 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. The nuclear pAkt S473 levels were quantified ( e ). N=30 cells from representative experiments of three repeats. f-g , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before processing for PLA between p53 and pAkt S473 . The nuclear PLA foci were quantified ( g ). N=30 cells from representative experiments of three repeats. h-i , IF staining of pAkt S473 in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h with or without the presence of the PI3Kα inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM). The nuclear pAkt S473 levels were quantified ( i ). N=30 cells from representative experiments of three repeats. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

Article Snippet: The PI-PolyPIPosomes for MST were purchased from Echelon Biosciences, including PI PolyPIPosomes (#Y-P000), PI4,5P 2 PolyPIPosomes (#Y-P045), and PI3,4,5P 3 PolyPIPosomes (#Y-P039).

Techniques: Transfection, Staining

Journal: Cell Reports

Article Title: The Axonal Membrane Protein PRG2 Inhibits PTEN and Directs Growth to Branches

doi: 10.1016/j.celrep.2019.10.039

Figure Lengend Snippet: PRG2 Regulates pAKT and PI(3,4,5)P 3 in Dependence of PTEN (A) PRG2 rescues decreased pAKT-levels induced by PTEN overexpression. HEK293 cells were triple transfected with AKT-GFP , GFP-PTEN (or control vector), and Prg2-Flag/Prg2ΔC-Flag (or control vector). Cell lysates were analyzed with indicated antibodies. (B) Bars represent normalized pAKT(S473) over total AKT ratios across treatments ± SEM of n = 5 independent experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001 (one-way ANOVA with Bonferroni post hoc test). (C) PRG2 rescues the loss of PI(3,4,5)P 3 -accumulations induced by PTEN overexpression. U87MG cells transfected with PH-AKT-mRFP (red) and GFP , GFP-PTEN C124S , GFP-PTEN (green, insets), and Prg2-Flag (white, insets). Panels show single confocal sections. Arrowheads indicate PH-AKT foci at plasma membranes (PM). Scale bar: 10 μm. (D) Cells were scored for the presence of peripheral PH-AKT foci. Bars show the means ± SD of two independent experiments performed in duplicate, n ≥ 50. ∗∗ p < 0.01 (t test). (E) PRG2 protects from the loss of peripheral F-actin accumulations induced by PTEN overexpression. Cells were stained with Phalloidin (F-actin, red top panels), anti-GFP (for detection of GFP or GFP-PTEN, green bottom panels) and anti-FLAG antibodies (for detection of PRG2-FLAG or PRG2ΔC-FLAG, white insets in lower magnification). Panels show maximum intensity confocal projections. Arrowheads indicate peripheral F-actin foci. Scale bar: 10 μm. (F) Cells were scored for presence of peripheral F-actin foci. Bars show means ± SD of three independent experiments performed in duplicates, n ≥ 60. ∗∗ p < 0.01 (t test).

Article Snippet: di-C8-PI(3,4,5)P 3 , Echelon Biosciences , Cat#P-3908.

Techniques: Over Expression, Transfection, Plasmid Preparation, Staining

Journal: Cell Reports

Article Title: The Axonal Membrane Protein PRG2 Inhibits PTEN and Directs Growth to Branches

doi: 10.1016/j.celrep.2019.10.039

Figure Lengend Snippet: Prg2 Overexpression in ES-Cell-Derived Neurons Induces Axon Filopodia Involving PI3K/PI(3,4,5)P 3 (A) Induction of Prg2-Flag expression after doxycycline (DOX) application. Lysates of ES-cell-derived motor neurons (ESCMNs) treated with 2 μg/mL doxycycline for 24 h were analyzed using the indicated antibodies. (B) ESCMN culture scheme. (C) Images show representative examples of neurons stained with Phalloidin (F-actin) after Prg2 induction with or without the PI3K inhibitor LY294002 (15 μM, 2 h). Scale bar: 10 μm. Doxycycline-induced Prg2 expression results in an increase in the number of axon filopodia. The PRG2-induced effect is reversed by PI3K inhibition using LY294002. (D) Quantification of filopodia. Bars show the mean number of filopodia per neuron, and the average length of filopodia/neuron ± SEM of 2–3 independent experiments performed in triplicates, n ≥ 100. **p < 0.01, ∗∗∗ p < 0.001 (one-way ANOVA with Bonferroni post hoc test). (E) Prg2- induced ESCMNs cultured for 24 h in the presence or absence of LY294002 (15 μM). PRG2 increases the number of axons and axon branches in dependence of PI3K/PI(3,4,5)P 3 . Cells were stained with anti-tubulin antibody (green) and Phalloidin (red). Scale bar: 30 μm. (F) Quantification of axons per cell and axonal branching. Bars show the mean number of branches ± SEM of two to three independent experiments performed in triplicates, n ≥ 120. ∗∗p < 0.01, ∗∗∗ p < 0.001 (one-way ANOVA with Bonferroni post hoc test). See also Figure S4 .

Article Snippet: di-C8-PI(3,4,5)P 3 , Echelon Biosciences , Cat#P-3908.

Techniques: Over Expression, Derivative Assay, Expressing, Staining, Inhibition, Cell Culture