Journal: bioRxiv

Article Title: Dephosphorylation of 4EBP1/2 Induces Prenatal Neural Stem Cell Quiescence

doi: 10.1101/2023.02.14.528513

Figure Lengend Snippet:

Article Snippet: p-S6 S240/244 conjugated to Ax488 , Rabbit , D68F8 , 5018S , Cell Signaling Technologies , 1:400 (Flow cytometry), 1:400) (IHC).

Techniques: Flow Cytometry

Journal: Cell death & disease

Article Title: Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK.

doi: 10.1038/cddis.2010.86

Figure Lengend Snippet: Figure 3 Cell fate following obatoclax is determined by additional mechanisms other than apoptosis. (a) Obatoclax fails to induce loss in plasma membrane integrity. (left) H460 and H1975 NSCLC cells were treated at indicated doses of obatoclax for 72 h. H1975 cells were also treated with 20 ng/ml TNF-receptor-related apoptosis-inducing ligand (TRAIL). Viability of cells was assessed by ViaLight viability assay (Lonza Group Ltd.). Data are expressed as mean±S.D. (right) H460 and H1975 cells were treated as for ViaLight, however, this time toxicity of cells was assessed by ToxiLight cytotoxicity assay (Lonza Group Ltd.). Data are expressed as mean±S.D. (b) Inhibition of obatoclax-induced apoptosis by ZVAD.fmk fails to rescue cells. (left) H1975 cells were treated with 10 mM pan caspase inhibitor ZVAD.fmk for 30 min before treatment with indicated doses of obatoclax for 48 h. Viability of cells was assessed by ViaLight viability assay. Data are expressed as mean±S.D. (right). The effect of ZVAD on obatoclax- induced PARP cleavage was assessed by SDS–PAGE. GAPDH was used as a loading control. (c) Obatoclax induces significant processing of LC3. (left) H460 and H1975 cells were treated with 500 nM obatoclax for 48 h and the level of LC3 processing assessed by SDS–PAGE. (right) Quantification of the level of LC3 processing following obatoclax was achieved by densitometry analysis using ImageJ. Data are expressed as mean±S.D. (d) Obatoclax-induced LC3 processing is independent of BAX and BAK. (left) H460shBAX/BAK 4C cells were treated with 1 mM obatoclax for 48 h and the level of LC3 processing assessed by SDS–PAGE. GAPDH was used as a loading control. (right) Wild type and BAX/BAK DKO MEFs were treated with 500 nM obatoclax for 48 h and the level of LC3 processing assessed by SDS–PAGE. a-Tubulin served as a loading control. (e) Obatoclax induces de-phosphorylation of S6 kinase. H460 and H1975 cells were treated with 500 nM obatoclax for 48 h and the level of S6 kinase phosphorylation determined by SDS–PAGE and immunoblotting with a phospho-S6 kinase antibody that detects phosphorylation of thr389. GAPDH was used as a loading control

Article Snippet: Rabbit primary antibodies used were those raised against cleaved caspase 9, cleaved caspase 3, LC3B, beclin-1, Atg5, Atg7, BAX, BAK and phospho S6 kinase (thr389) (all from Cell Signaling Technology Inc., Danvers, MA, USA), BAK N-terminus (Millipore, Billerica, MA, USA) and a-tubulin (Abcam, Cambridge, MA, USA).

Techniques: Clinical Proteomics, Membrane, Viability Assay, Cytotoxicity Assay, Inhibition, SDS Page, Control, De-Phosphorylation Assay, Phospho-proteomics, Western Blot

Journal: Hormones (Athens, Greece)

Article Title: Anticancer effects of metformin on neuroendocrine tumor cells in vitro.

doi: 10.14310/horm.2002.1517

Figure Lengend Snippet: Figure 3. Effect of metformin on PI3K/AKT/mTORC1 sig- naling in neuroendocrine. Human pancreatic BON1 (A, B), bronchopulmonary NCI-H727 (C, D) and midgut GOT1 (E, F) neuroendocrine tumor cells were treated with indicated concentrations of metformin for 24 and 48 hrs, respectively. Subsequently the expression of pAKT, AKT, pEBP1, EBP1, IGFR (A, C and E) as well as pP70S6K, P70S6K, pS6, S6 (B, D and F) and ą-actin loading control was evaluated by Western blot analysis. A representative blot out of 3 independently per- formed experiments is shown.

Article Snippet: After blocking in 2% non-fat dried milk, the membranes were incubated overnight in appropriate dilutions of antibodies against pAkt (Ser 473) (#4060), Akt (#2920), pERK (Thr202/Tyr204) 1/2 (#4370), pP70S6K (Thr389) (#9234), P70S6K (#9202), p4EBP1 (Ser65) (#9451), 4EBP1 (#9644), pS6 (Ser235/6 (#4858) and Ser240/4 (#5364)), S6 (#2317), pGSK3 (Ser21/9) (#9331), GSK3 (#9315), IGFR (#3027), EGFR (#4267), PARP (#9542) (all from Cell Signaling, Danvers, MA), Erk 1/2 (06-182; Millipore).

Techniques: Expressing, Control, Western Blot

Journal: Hormones (Athens, Greece)

Article Title: Anticancer effects of metformin on neuroendocrine tumor cells in vitro.

doi: 10.14310/horm.2002.1517

Figure Lengend Snippet: Figure 4. Effect of metformin and rapamycin on IGF1-induced signaling in neuroendocrine tumor cells. Human pancreatic BON1 (left panel) and bronchopulmonary NCI-H727 (right panel) neuroendocrine tumor cells were pretreated with indi- cated concentrations of metformin or rapamycin for 1 hr prior to induction with IGF1 for 24 hrs. The expression of pIGFR, IGFR, pAKT, AKT, pS6, S6 and ą-actin loading control was evaluated by Western blot analysis. A representative blot out of 3 independently performed experiments is shown.

Article Snippet: After blocking in 2% non-fat dried milk, the membranes were incubated overnight in appropriate dilutions of antibodies against pAkt (Ser 473) (#4060), Akt (#2920), pERK (Thr202/Tyr204) 1/2 (#4370), pP70S6K (Thr389) (#9234), P70S6K (#9202), p4EBP1 (Ser65) (#9451), 4EBP1 (#9644), pS6 (Ser235/6 (#4858) and Ser240/4 (#5364)), S6 (#2317), pGSK3 (Ser21/9) (#9331), GSK3 (#9315), IGFR (#3027), EGFR (#4267), PARP (#9542) (all from Cell Signaling, Danvers, MA), Erk 1/2 (06-182; Millipore).

Techniques: Expressing, Control, Western Blot