Journal: Neural Regeneration Research

Article Title: PTEN inhibitor bisperoxovanadium protects against noise-induced hearing loss

doi: 10.4103/1673-5374.358606

Figure Lengend Snippet: bpV prevents noise-induced reductions in PI3K-Akt signaling in OHCs. (A, B) Representative images showing the reduced levels of PI3K-p85α and -p110α (red, Alexa Fluor 594) in OHCs after noise exposure, as detected by immunofluorescence staining. Pretreatment with bpV protected against these effects. Scale bars: 20 μm. (C, D) Quantification of PI3K-p85α and -p110α levels in OHCs in noise (PTS) and noise + bpV (PTS + bpV) groups. (E, F) Representative images and quantification analyses showed that p-Akt levels were reduced in OHCs after noise exposure. (G) mRNA expression levels of PI3K and Akt were detected by real-time PCR in control, noise, and noise + bpV groups. (H) Protein expression of PI3K-p85α, -p110α, p-Akt and Akt were determined by western blotting. (I–K) Quantification of PI3K-p85α and -p110α relative to GAPDH and of p-Akt/Akt. Expression of p110α decreased after noise exposure compared with that in the control group. There was no change in total Akt level between the three groups. Data are presented as the mean ± SD. The experiment was repeated three times. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired Student’s t -test). bpV: Bisperoxovanadium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IHC: inner hair cell; OHC: outer hair cell; p-Akt: phospho-Akt; PCR: polymerase chain reaction; PI3K: phosphoinositide-3 kinase; PTS: permanent threshold shift.

Article Snippet: The primary antibodies were monoclonal rabbit anti-PTEN (Cell Signaling Technology, Cat# 9188, RRID: AB_2253290), monoclonal rabbit anti-PI3K-p110α (Cell Signaling Technology, Cat# 4249, RRID: AB_2165248), polyclonal rabbit anti-PI3K-p85α (Cell Signaling Technology, Cat# 4292, RRID: AB_329869), monoclonal rabbit anti-phospho-Akt (Ser 473) (Cell Signaling Technology, Cat# 4060, RRID: AB_2315049) and polyclonal rabbit anti-4 hydroxynonenal (4HNE; Abcam, Cambridge, UK, Cat# ab46545, RRID: AB_722490).

Techniques: Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

doi: 10.4103/1673-5374.355978

Figure Lengend Snippet: Argon preconditioning reduces rotenone-induced protein expression of Bax and increases Bcl-2, but the effect is abolished by OxPAPC. (A, C) Densitometric analysis of western blots for Bax (A) and Bcl-2 (C) protein expression. Between-group comparisons were performed with a one-way analysis of variance using the post hoc Holm Sidak test ( n = 6; mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001). (B, D) Representative western blot images of six independent experiments that showed similar results demonstrating the influence of rotenone, argon preconditioning, and inhibition with OxPAPC on Bax (B) and Bcl-2 (D).

Article Snippet: After protein transfer to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Schwalbach, Germany), the membranes were blocked with 5% skim milk in Tween20/PBS (TBST) or BSA (bovine serum albumin) and incubated in the recommended dilution of protein-specific antibody (p44/42 MAP kinase (phospho-ERK1/2 [extracellular-signal regulated kinase]) (Thr202/Tyr204), rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 9101, RRID: AB_331646; phospho-NF-κB (Ser536) (93H1), rabbit, 1:1000, Cell Signaling Technology, Cat# 3033, RRID: AB_331284; phospho-Akt (protein kinase B) antibody (Ser473), rabbit, 1:1000, Cell Signaling Technology, Cat# 9271, RRID: AB_329825; Bax antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2772, RRID: AB_10695870; Bcl-2 antibody, rabbit, 1:1000, Cell Signaling Technology, Cat# 2876, RRID: AB_2064177; and cleaved caspase-3 antibody (Asp175), rabbit, 1:500, Cell Signaling Technology, Cat# 9661, RRID: AB_2341188; NF-E2-related factor 2 (Nrf2) antibody [nuclear factor erythroid 2-related factor 2, phosphor-S40], rabbit, 1:60000, Abcam, Cat# ab76026, RRID: AB_1524049).

Techniques: Expressing, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia

doi: 10.1101/2023.05.29.542723

Figure Lengend Snippet: Caspase-2 regulates pathways that maintain stemness in NPM1c+ cells. (A) Reverse phase protein array (RPPA) analysis was carried out on OCI-AML2 ( NPM1c+ ) parental and ΔC2 cells. Signal intensities were normalized and filtered as described in Materials and Methods. The heat map shows Z-scores of three biological replicates of parental NPM1c+ cells and each ΔC2 clone with q > 0.01. Upregulated proteins are shown in red and down regulated proteins are shown in blue. Proteins in the mTORC1/AKT pathway are labeled in blue and proteins in the Wnt signaling pathway are labeled in green. (B) OCI-AML-2 ( NPM1 wt) and OCI-AML-3 ( NPM1c +) parental or ΔC2 cells were left untreated or treated with IL-2 for 16 h. Lysates were harvested and immunoblotted for phosphorylated AKT (p-AKT) and total AKT. Actin was used as the loading control. A spacer lane was cropped from the actin and AKT blots for alignment purposes. (C) NPM1wt and NPM1 c+ parental or ΔC2 cells were treated with or without IGF-1 (10 ng/mL) for 16 h. Lysates were harvested and immunoblotted for p-AKT and total AKT. Actin was used as the loading control. (D) NPM1wt and NPM1 c+ parental or ΔC2 cells were treated with or without Wnt (5.0 nM) for 16 h. Lysates were harvested and immunoblotted for β- catenin (active) and phosphorylated β-catenin (inactive). Actin was used as the loading control. The panels for each protein are from the same immunoblot with irrelevant lanes cropped out. Experiments shown in (B–D) are representative of 2–3 independent experiments.

Article Snippet: The following antibodies were used: anti-NPM1 (clone FC82291; sigma-aldrich); anti-caspase-2 (clone 11B4; EMD Millipore); anti-fibrillarin (C13C3; Cell Signaling Technology); anti-BID (AF860; R&D systems); anti-caspase-3 (9662; Cell Signaling Technology); anti-caspase-3 cleaved (9661; Cell Signaling Technology); anti-Actin (clone 4; Fisher Scientific); anti-phospho-AKT (4060; Cell Signaling Technology), anti-AKT (4691; Cell Signaling Technology); anti-β-catenin (active) (8814; Cell Signaling Technology); anti-β-catenin (inactive) (961; Cell Signaling Technology); anti-B23 (172-6195; Fisher-scientific); APC/Cy7 anti-human CD34 (clone 581; BioLegend); APC anti-human CD14 (clone 63D3; BioLegend).

Techniques: Protein Array, Labeling, Western Blot