Journal: Molecular neurobiology

Article Title: Transcranial Direct-Current Stimulation Regulates MCT1-PPA-PTEN-LONP1 Signaling to Confer Neuroprotection After Rat Cerebral Ischemia-Reperfusion Injury.

doi: 10.1007/s12035-022-03051-7

Figure Lengend Snippet: Fig. 5 The downregulation of PTEN enhances LONP1 expres- sion following OGD injury. a Western blotting analyses indicated that PTEN regulates the expression of LONP1 within neurons. Primary neurons were treated with siRNA of PTEN for 72 h, after which Western blotting was conducted (n = 6/ group, *p < 0.05 vs. sham, #p < 0.05 vs. sham, Student’s t-test). b Western blotting indicated that LONP1 had no impact on PTEN expres- sion within neurons. Neurons were treated with bortezomib for 72 h, after which Western blotting was conducted (n = 6/ group, *p < 0.05 vs. sham, Student’s t-test). c Western blotting conducted at 3 and 6 h following reoxygenation indicated that neurons exhibited PTEN upregulation in cultured cortical neurons (n = 6/group, F (4, 25) = 8.37, *p < 0.05 vs. sham 3 h, #p < 0.05 vs. sham 6 h, one-way ANOVA). d West- ern blotting revealed increased PTEN levels within the rat cer- ebral cortex following cerebral ischemia–reperfusion injury. Peri-infarct cortical tissue was collected for analysis at 3 and 6 h following occlusion for 1 h (n = 6/group, F (4, 25) = 4.72, *p < 0.05 vs. sham 3 h, #p < 0.05 vs. sham 6 h, one-way ANOVA)

Article Snippet: The PTEN siRNA (siRNApten) and non-targeting control siRNA (NsiRNA) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Western Blot, Expressing, Cell Culture

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells.

doi: 10.1016/j.biopha.2022.113366

Figure Lengend Snippet: Fig. 1. Production of GRA16-stable HCT116 cells, expression of signaling molecules involved in hTERT transcription, and decreases in the expression and phos phorylation of hTERT in GRA16-stable cells. (A) PCR results for vector gene (pBABE-HA II) or gra16 gene (pBABE-HA II-gra16) using primers for vector (Vp) and GRA16 (Gp). (B) HA-tagged GRA16 expressions in experimental groups (control, vector, and GRA16). (C) Telomere length analyzed by qPCR using telomere-specific primers (Tel1/2) and single-copy gene primers (36B4u/d). Their ratio (T/S) was used for the calculation of telomere length (n = 16). (D) Expressions of signaling molecules (HAUSP, PP2A-B55, p-PTEN/PTEN, and p-NF-κB p65/NF-κB p65) in experimental groups (n = 3). (E) Expressions of signaling molecules involved in the transcription and activation of hTERT (p-P53/P53, p-AKT(S473)/AKT, p-AKT(T308)/AKT, p-STAT3/STAT3, E2F1, and c-Myc) (n = 3). (F) mRNA expressions of shelterin complex factors and hTERT transcriptional factors in experimental groups (n = 3). (G) hTERT expressions and p-hTERT/hTERT levels in experimental groups (n = 3). Data represent mean ± standard deviation (SD). Each expression value of protein or mRNA of signaling molecules in the control group was fixed at “1,” after which the relative expression of each signaling molecule in vector and GRA16 groups was calculated. * significant difference between control and GRA16 groups (P < 0.05); † significant difference between vector and GRA16 groups (P < 0.05).

Article Snippet: GRA16-expressing HCT116 cells (GRA16-stable cells) were seeded in 6-well plates at 2 × 105 cells/well and incubated for 24 h. Then, the cells were transfected with control siRNA (Santa Cruz Biotechnology), PTEN siRNA (Santa Cruz Biotechnology), or PP2A-B55 siRNA (Santa Cruz Biotechnology) using the Lipofectamine 3000 transfection kit (Life Technologies) and Opti-MEM medium (Life Technologies).

Techniques: Expressing, Plasmid Preparation, Control, Activation Assay, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells.

doi: 10.1016/j.biopha.2022.113366

Figure Lengend Snippet: Fig. 2. Increase in the nuclear localization of PTEN and decrease in the nuclear localization of NF-κB p65 in HCT116 stable cells. (A) Immunofluorescence images illustrated the increased translocation of PTEN into the nuclei of HCT116 GRA16 cells. Scale bars = 50 µm. Yellow arrows indicate PTEN translocated into the nuclei of HCT116 GRA16 cells (n = 3). (B) Western blot images of PTEN expression in the whole-cell (Whole), cytoplasmic (Cyto), and nuclear fractions (Nuc) of HCT116 stable cells. PTEN levels in these fractions are presented to those of housekeeping proteins (β-actin and lamin B; n = 3). (C) Immunofluorescence images presenting the decreased nuclear expression of NF-κB p65 in HCT116 GRA16 cells. Scale bars = 50 µm. White arrows indicate NF-κB p65 translocated to the nucleus of HCT116 GRA16 cells (n = 3). (D) Western blot images of NF-κB p65 expression in Whole, Cyto, and Nuc lysates in HCT116 stable cells. NF-κB p65 levels in these fractions are presented relative to those of housekeeping proteins (β-actin and lamin B; n = 3). Data are presented as the mean ± SD. Results in bar graphs present the relative protein expression (PTEN or NF-κB p65) in vector and GRA16 cells relative to the value in control cells, which was fixed at “1.” * Significant difference between the control and GRA16 groups (P < 0.05); † Significant difference between the vector and GRA16 groups (P < 0.05).

Article Snippet: GRA16-expressing HCT116 cells (GRA16-stable cells) were seeded in 6-well plates at 2 × 105 cells/well and incubated for 24 h. Then, the cells were transfected with control siRNA (Santa Cruz Biotechnology), PTEN siRNA (Santa Cruz Biotechnology), or PP2A-B55 siRNA (Santa Cruz Biotechnology) using the Lipofectamine 3000 transfection kit (Life Technologies) and Opti-MEM medium (Life Technologies).

Techniques: Immunofluorescence, Translocation Assay, Western Blot, Expressing, Plasmid Preparation, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells.

doi: 10.1016/j.biopha.2022.113366

Figure Lengend Snippet: Fig. 5. Identification of signaling pathway to induce hTERT inactivation in GRA16 stable cells using inhibitors of PTEN and PP2A-B55 (SF1670 and LB-100, respectively). (A) Western blot images of GRA16/HAUSP/PTEN/AKT(S473) signaling pathway molecules when GRA16-stable cells were treated with SF1670, LB- 100, or both inhibitors and their relative protein expression (n = 3). (B) Western blot images of GRA16/PP2A-B55/p-NF-κB p65/p-AKT(T308) signaling pathway molecules when GRA16-stable cells were treated with SF1670, LB-100, or both inhibitors and their relative protein expressions (n = 3). (C) Western blot images of hTERT and p-hTERT and their relative protein expressions (n = 3). (D) Relative mRNA expressions of shelterin complex factors and hTERT transcriptional factors responding to inhibitors of PTEN and PP2A-B55 (SF1670 and LB-100, respectively) in GRA16 cells when the expression value of each molecule in nontreated GRA16 cells (non) was set to “1” (n = 3). (E) Relative mRNA expressions of cell cycle-related factors and apoptosis-related factors responding to inhibitors of PTEN and PP2A-B55 (SF1670 and LB-100, respectively) (n = 3). Relative expressions of proteins or mRNAs were calculated by comparing each expression value in GRA16 cells treated with each inhibitor when their expression value in nontreated GRA16 cells (non) was set to “1.” Data in this figure represent mean ± SD. ‡ significant difference between nontreated cells and SF1670- and/or LB-100-treated cells (P < 0.05).

Article Snippet: GRA16-expressing HCT116 cells (GRA16-stable cells) were seeded in 6-well plates at 2 × 105 cells/well and incubated for 24 h. Then, the cells were transfected with control siRNA (Santa Cruz Biotechnology), PTEN siRNA (Santa Cruz Biotechnology), or PP2A-B55 siRNA (Santa Cruz Biotechnology) using the Lipofectamine 3000 transfection kit (Life Technologies) and Opti-MEM medium (Life Technologies).

Techniques: Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells.

doi: 10.1016/j.biopha.2022.113366

Figure Lengend Snippet: Fig. 6. Changes in hTERT-related signaling molecules in GRA16-stable cells transfected with PTEN siRNA or PP2A-B55 siRNA. (A) Western blot images and their relative protein expressions for GRA16/HAUSP/PTEN/AKT(S473) signaling pathway molecules and hTERT transcription factors in PTEN siRNA- or PP2A-B55 siRNA- transfected GRA16 cells (n = 3). (B) Western blot images and their relative protein expressions for GRA16/PP2A-B55/NF-κB p65/AKT(T308) signaling pathway molecules in PTEN siRNA- or PP2A-B55 siRNA-transfected GRA16 cells (n = 3). (C) Western blot images and their relative protein expressions for hTERT and p- hTERT in PTEN siRNA- or PP2A-B55 siRNA-transfected GRA16 cells (n = 3). The expression value of each molecule in the nontransfection group (mock) was set to “1,” and then the relative expression of each molecule in GRA16 cells transfected with PTEN siRNA or PP2A-B55 siRNA was calculated. Data represent mean ± SD. * significant difference at P < 0.05 between control siRNA transfection and PTEN siRNA or PP2A-B55 siRNA transfection.

Article Snippet: GRA16-expressing HCT116 cells (GRA16-stable cells) were seeded in 6-well plates at 2 × 105 cells/well and incubated for 24 h. Then, the cells were transfected with control siRNA (Santa Cruz Biotechnology), PTEN siRNA (Santa Cruz Biotechnology), or PP2A-B55 siRNA (Santa Cruz Biotechnology) using the Lipofectamine 3000 transfection kit (Life Technologies) and Opti-MEM medium (Life Technologies).

Techniques: Transfection, Western Blot, Expressing, Control