Journal: The Journal of Experimental Medicine

Article Title: Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells

doi:

Figure Lengend Snippet: Resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis. (A) and (B) Flow cytometry analysis of staining by annexin V of Jurkat cells treated with Ad (10 PFU/ml), GrB (1 μg/ml), and a combination of GrB and Ad for 2 h (A) or 24 h (B) at 30°C. The data are means ± SD of results obtained in five independent experiments. The asterisks indicate a statistically significant difference between wild-type and Bak-deficient cells ( P < 0.05, Mann-Whitney U ). (C) GrB-mediated cleavage of PARP and DFF45/ICAD in wild-type, but not in Bak-deficient cells. Wild-type and Bak-deficient cells were treated with Ad, GrB, or a combination of GrB and Ad, as described previously. The cell extracts were resolved on SDS/PAGE and immunoblotted with anti-PARP mAb or anti-DFF45/ICAD Ab. (D) and (E) Lack of mitochondrial apoptotic events in Bak-deficient Jurkat cells treated with GrB. After 2 h of treatment with GrB and Ad, as described previously, the cells were assessed by flow cytometry for mitochondrial staining with CMXRos or NAO. Staining with CMXRos (100 nM) served to assess changes in mitochondria permeability transition; staining with NAO (100 nM) served to assess loss in mitochondrial cardiolipin. (F) and (G) Susceptibility of Bak-deficient Jurkat cells to TRAIL or taxol. Wild-type or Bak-deficient Jurkat cells were treated with TRAIL (100 ng/ml) or taxol (10 μg/ml) for 16 h. The cells were then analyzed by flow cytometry for staining by annexin V or propidium iodide. Percentage of apoptotic cells are indicated for TRAIL.

Article Snippet: We also used anti-Bid Ab from BioVision and from Santa Cruz Biotechnology, Inc.; anticaspase-3 was from BD PharMingen; anti-poly-(ADP-ribose) polymerase (PARP) mAb (C2.10) and Cbz-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) were from Enzyme System; rabbit anti-DNA fragmentation factor (DFF)45/inhibitor of caspase-activate DNase (ICAD) Ab was from ProSci.

Techniques: Flow Cytometry, Staining, MANN-WHITNEY, SDS Page, Permeability

Journal: International Dental Journal

Article Title: High-Glucose Microenvironment Accelerates Malignant Progression Via O-GlcNAcylation in Oral Squamous Cell Carcinoma

doi: 10.1016/j.identj.2025.103897

Figure Lengend Snippet: The effects of OGT on cell proliferation, metastasis, and apoptosis partially depend upon the PI3K/AKT/mTOR signalling pathway in OSCC cells. (A-C) Levels of PI3K, AKT, mTOR, p-PI3K, p-AKT, p-mTOR, OGT, and O-GlcNAcylation proteins were assessed in OSCC cells exposed to a medium with different glucose concentrations and OGT knockdown. n = three independent experiments. Data were presented as the mean ± SD. P values were determined by one-way ANOVA. * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: Antibodies against N-cadherin (N-cad) (1:1000 for WB, HY- P80238 ), E-cadherin (E-cad) (1:1000 for WB, P80113), vimentin (1:1000 for WB, HY- P80371 ), Bcl2 (1:1000 for WB, P80566 ), Bax (1:1000 for WB, P80028 ), PI3K (1:1000 for WB, HY- P80867 ), and phosphorylated P13K (p-PI3K) (1:1000 for WB, HY- P80846 ) were purchased from MedChemExpress; antibodies against AKT (1:1000 for WB, 4691T) and p-AKT (1:1000 for WB, 13038T) were purchased from Cell Signaling Technology; antibodies against OGT (1:5000 for WB, 1:250 for IHC, 66823-1-Ig), mammalian target of rapamycin (mTOR) (1:5000, 66888-1-Ig), p-mTOR (1:5000, 67778-1-Ig), and β-Actin (1:20,000 for WB, 66009-1-Ig) were obtained from Proteintech; and the O-GlcNAc antibody (1:1000 for WB, 1:200 for IHC, MA1-072) was purchased from Invitrogen.

Techniques: Knockdown

Journal: The Journal of Neuroscience

Article Title: Differential Control of Thrombospondin over Synaptic Glycine and AMPA Receptors in Spinal Cord Neurons

doi: 10.1523/JNEUROSCI.5247-12.2013

Figure Lengend Snippet: Differential effects of TSP-1 on the amount of excitatory and inhibitory receptors at synapses. A, Receptor IR in control conditions (top) or after 1 h TSP-1 treatment (bottom). Double immunodetection of receptors (green) and their corresponding scaffolding molecules (red): GluA2-AMPAR and PSD95 (left), GluA1-AMPAR and PSD95 (center) or α1-GlyR and gephyrin (right). Scale bar, 5 μm. B–F, Quantification of the fluorescence associated with the indicated receptors. B, Variation of receptor IR (% of control) after 10 or 60 min of TSP-1 treatment (n = 60, ANOVA and Tukey test). C, Variation of the indicated receptor IR (% of control) after application of TSP-1 (60 min) in the presence of TTX (n = 40–60, t test). D, Variation of the GlyR-IR after application of TSP-1 (60 min) alone (T) or in the presence of CNQX (T+CNQX) or d-AP5 (T+d-AP5). Drugs were added 10 min before TSP-1 (n = 40–60, t test). E, F, Normalized IR associated with synaptic GluA2 (E) and GlyR (F) in control conditions (Ctr) or after incubation with TSP-1 (T), anti-β1 integrin antibody (B1), anti-β1 antibody before TSP-1 (B1+T), anti-β3 integrin antibody (B3) or anti-β3 antibody before TSP-1 (B3+T). Treatments lasted 10 min in each case (n = 15–31, t test). G1, G2, Normalized fluorescence of phalloidin colocalized with PSD-95-IR (G1) or gephyrin-IR (G2) in control conditions (control) or after incubation with TSP-1 for the indicated times (n = 86–181, ANOVA and Tukey test). H1, H2, Quantification of IR associated with synaptic GluA1 (H1) or GluA2 (H2) in control conditions or after treatment with TSP-1 (TSP, 10 min), jasplakinolide (Jas), or jasplakinolide and TSP-1 (Jas+TSP) (n = 20–47, t test). All values are the mean ± SEM. Ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Immunodetection was performed using anti-α-GlyR (mAb4a or mAb2b, Synaptic Systems), anti-α1-GlyR ( Charrier et al., 2010 ), anti-GluA1 (Alomone), anti-GluA2 (BD PharMingen or Synaptic Systems), anti-gephyrin (mAb7a, Synaptic Systems), anti-PSD95 (6G6–1C9, Abcam), anti-Piccolo (kindly provided by E.D.

Techniques: Immunodetection, Scaffolding, Fluorescence, Incubation