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: Fasting produces antidepressant-like effects via activating mammalian target of rapamycin complex 1 signaling pathway in ovariectomized mice

doi: 10.4103/1673-5374.367928

Figure Lengend Snippet: Fasting increases protein expression in the mTORC1 signaling pathway in the HP and PFC of ovariectomized mice. (A) Representative western blots for the mTORC1 signaling pathway in the HP. (B–J) Relative protein expression of mTORC1 (B), BDNF (C), AKT (D), pAKT/AKT (E), ERK (F), pERK/ERK (G), p70S6 (H), S6 (I), and PSD95 (J) in the HP. (K) Representative western blots for the mTORC1 signaling pathway in the PFC. (L–T) Relative protein expression of mTORC1 (L), BDNF (M), AKT (N), pAKT/AKT (O), ERK (P), pERK/ERK (Q), p70S6 (R), S6 (S), and PSD95 (T) in the PFC. Values are represented as the mean ± SEM ( n = 4–9 per group). * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s honestly significant difference test). AKT: Protein kinase B; BDNF: brain-derived neurotrophic factor; ERK: extracellular signal-regulated protein; F: fasting; HP: hippocampus; mTORC1: mammalian target of rapamycin complex 1; OV: ovariectomy; p70S6: p70 ribosomal S6 kinase; pAKT: phosphor-AKT; pERK: phosphor-ERK; PFC: prefrontal cortex; R: rapamycin; S6: small ribosomal protein 6; PSD95: postsynaptic density protein 95; Sham: sham treatment.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4°C: estrogen receptor α (rabbit polyclonal, 1:1000; Affinity, Cincinnati, OH, USA, Cat# AF6058, RRID: AB_2834976), estrogen receptor β (rabbit polyclonal, 1:1000; Affinity, Cat# AF6469, RRID: AB_2835288), BDNF (rabbit polyclonal, 1:800; Santa Cruz Bio, Dallas, TX, USA, Cat# sc546, RRID: AB_630940), AKT (rabbit polyclonal, 1:1000; CST, Danvers, MA, USA, Cat# 9272, RRID: AB_329827), phosphor-AKT (pAKT; rabbit polyclonal, 1:1000; CST, Cat# 9271, RRID:AB_329825), ERK (rabbit polyclonal, 1:1000; CST, Cat# 9102, RRID: AB_330744), phosphor-ERK (pERK; rabbit polyclonal, 1:1000; CST, Cat# 9101, RRID: AB_331646), mTORC1 (rabbit monoclonal, 1:1000; CST, Cat# 2587, RRID:AB_2261091), p70S6 (rabbit polyclonal, 1:1000; CST, Cat# 9202, RRID:AB_331676), S6 (rabbit monoclonal, 1:1000; CST, Cat# 2217, RRID: AB_331355), 4E-binding protein 1 (4EBP1; rabbit polyclonal, 1:1000; CST Cat# 9452, RRID: AB_331692), PSD95 (mouse monoclonal, 1:1000; Abcam, Cambridge, UK, Cat# ab192757, RRID: AB_2750929), and β-actin (mouse monoclonal, 1:2000; Transgen Biotech, Beijing, China, Cat# HC201, RRID: AB_2860007).

Techniques: Expressing, Western Blot, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: SETDB1 expression is associated with PI3K/AKT signaling and epithelial-mesenchymal transition activation in multiple myeloma cells. (A) Gene set enrichment analysis illustrating that SETDB1 expression is positively associated with the PI3K/AKT/mTOR signaling pathway. Western blot analysis in (B) U266 and (C) RPMI-8226 cells. *** P<0.001 vs. SETDB1-NC. ## P<0.01, ### P<0.001 vs. shSETDB1-NC. SETDB1, SET domain bifurcated histone lysine methyltransferase 1; NC, negative control; OE, overexpression; sh, short hairpin RNA; N-cad, N-cadherin; FDR, false discovery rate; NES, normalized enrichment score; p, phosphorylated.

Article Snippet: LY294002 (cat. no. #9901; Cell Signaling Technology, Inc.), a specific inhibitor of the PI3K/AKT pathway, were diluted to a concentration of 10 µg/ml.

Techniques: Expressing, Activation Assay, Western Blot, Negative Control, Over Expression, shRNA

Journal: Experimental and Therapeutic Medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: PI3K/AKT signaling and epithelial-mesenchymal transition contribute to SETDB1-induced lenalidomide resistance in multiple myeloma. (A) Flow cytometry analysis of apoptosis of U266 and RPMI-8226 cells after treatment with LY294002 and SETDB1 overexpression. (B) Statistical analysis of apoptosis in each group. ** P<0.01, *** P<0.001 vs. SETDB1-NC; ### P<0.001 vs. LY294002 + SETDB1-NC. (C) IC 50 values of lenalidomide were determined by Cell Counting Kit-8 assay. LY294002 significantly reduced the IC 50 value of lenalidomide, whereas SETDB1 overexpression attenuated this effect. (D) Western blot analysis revealed that the expression of N-cadherin and vimentin, as well as the phosphorylation of PI3K and AKT, were inhibited by LY294002; these effects were counteracted by SETDB1 overexpression. *** P<0.001 vs. SETDB1-NC; # P<0.05, ## P<0.01 and ### P<0.001 vs. LY294002 + SETDB1-NC. SETDB1, SET domain bifurcated histone lysine methyltransferase 1; NC, negative control; OE, overexpression; IC 50 , half maximal inhibitory concentration; N-cad, N-cadherin; p, phosphorylation.

Article Snippet: LY294002 (cat. no. #9901; Cell Signaling Technology, Inc.), a specific inhibitor of the PI3K/AKT pathway, were diluted to a concentration of 10 µg/ml.

Techniques: Flow Cytometry, Over Expression, Cell Counting, Western Blot, Expressing, Negative Control, Concentration Assay