Journal: Journal of Neuroscience

Article Title: Pulse Inhibition of Histone Deacetylases Induces Complete Resistance to Oxidative Death in Cortical Neurons without Toxicity and Reveals a Role for Cytoplasmic p21waf1/cip1 in Cell Cycle-Independent Neuroprotection

doi: 10.1523/jneurosci.3200-07.2008

Figure Lengend Snippet: Figure9. p21actsinthecytoplasmtoprotectneuronsfromoxidativestress-induceddeath. A,Westernblotanalysistodetectrelativelevelsofp21innuclearandcytoplasmicfractionsfrom primarycorticalneuronalculturestreatedwithTSA(0.66M)inthepresenceorabsenceofHCA (5mM).ControlisnoTSAtreatment.RelativelevelsofGAPDHorNeuNandHistoneH3areshown to indicate purity of cytoplasmic or nuclear fractions, respectively. Antibodies against p21, GAPDH,NeuN,andHistoneH3wereused(seeMaterialsandMethods).B,Westernblotanalysis to detect relative levels of GFP, p21-GFP fusion protein or p21NLS-GFP fusion protein in cytoplasmic (C) and nuclear (N) fractions from HT22 murine hippocampal cells stably trans- fected with pEGFP, pEGFP-p21, or pEGFP-p21-NLS and treated with or without HCA (5 mM). Antibodies against p21 (top) or GFP (middle) were used (see Materials and Methods). The antibody for p21 does not detect the p21NLS-GFP fusion protein because the epitope this monoclonalantibodyrecognizesislocatedwithinthedeletednuclearlocalizationsignal(NLS). However, in addition to p21-GFP, this antibody detects endogenous p21, which is unchanged withHCAtreatment.C,GraphshowingviabilityofpEGFP-(darkgray),pEGFP-p21-(lightgray), and pEGFP-p21-NLS- (medium gray) transfected HT22 cells, as determined using the MTT assay,aftertreatmentwithincreasingconcentrationsofHCA(2.5–7.5mM)for24h.Graphbars depict mean SD. *Significant protection by p21 and p21-NLS relative to GFP control, p 0.001, by two-way ANOVA followed by Bonferroni posttests.

Article Snippet: Nonspecific binding was inhibited by incubation in Tris-buffered saline with Tween 20 (TBST: 50 mM Tris-HCl, pH 8.0, 0.9% NaCl, and 0.1% Tween 20) containing 5% nonfat milk for at least 1.5 h. Primary antibodies against p21 (BD Biosciences, San Jose, CA), p15 (Santa Cruz Biotechnology, Santa Cruz, CA), p16 (BD Biosciences), p27 (BD Biosciences), p57 (Millipore, Billerica, MA), pRb (BD Biosciences), GFP (Invitrogen), histone H4 (Millipore), acetyl histone H4 (Millipore), histone H3 (Millipore), phospho-JNK (Cell Signaling, Danvers, MA), total JNK (Cell Signaling), GAPDH (Millipore), NeuN (Millipore), -tubulin (Sigma-Aldrich), and HA (Sigma-Aldrich) were diluted in TBST containing 5% milk overnight at 4°C followed by incubation with respective horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) for 2 h at room temperature.

Techniques: Stable Transfection, Transfection, MTT Assay, Control

Journal: bioRxiv

Article Title: HAUSP Stabilizes SOX2 through Deubiquitination to Maintain Self-renewal and Tumorigenic Potential of Glioma Stem Cells

doi: 10.1101/2021.06.09.447550

Figure Lengend Snippet: (A) Immunoblot analyses of differentiated cell markers TUJ1 (a neuronal marker) and GFAP (an astrocyte marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and harvested for immunoblot analyses with specific antibodies against TUJ1, GFAP, HAUSP, and GAPDH (loading control). (B) Quantitative PCR (RT-PCR) analyses of TUJ1 and GFAP mRNA levels in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for the indicated days, and harvested for RT-PCR analyses. Student t test was used to assess the significance. ***, p<0.001. ( C and D ) Immunofluorescent staining of TUJ1 (a neuronal marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and immunostained with a specific antibody against TUJ1 (in green) and counterstained with DAPI to indicate nuclei (in blue). Scale bar, 30 µm. Quantifications of TUJ1-positive cells were shown (D). Student t test was used to assess the significance. ns, no significant difference; ***, p<0.001. ( E and F ) Immunofluorescent staining of GFAP (an astrocyte marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and immunostained with a specific antibody against GFAP (in green) and counterstained with DAPI to indicate nuclei (in blue). Scale bar, 30 µm. Quantifications of GFAP-positive cells were shown (F). Student t test was used to assess the significance. ns, no significant difference; ***, p<0.001.

Article Snippet: Specific antibodies against HAUSP (Bethyl, A300-033A), SOX2 (Bethyl, A201-741A; or Santa Cruz, SC-365964), OLIG2 (R&D System, AF2418), GFAP (Biolegend, 644702), TUJ1 (Biolegend, 801201), MAP2 (Biolegend, 801801), GAPDH (R&D System, MAB5718), HA (Sigma, 11867423001), α-Tubulin (Sigma, T6199), Flag (Sigma, F1804), Ubiquitin (Biolegend, 646302), Cleaved Caspase 3 (Cell Signaling, 9661), Cleaved PARP (Cell Signaling, 5625), and anti-Flag-agarose (Sigma, A2220) were used for immunoblot and/or immunoprecipitation.

Techniques: Western Blot, Marker, Expressing, Control, Transduction, Infection, Cell Culture, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 3. Salicylate affects multiple cell types and has diverse effects. (A) U2OS osteosarcoma cells and (B) MCF-7 breast cancer cells were grown in complete media for 48 h in the presence of salicylate (lane 2, (A,B), respectively), the synthetic activator A76966 (lane 3, (A,B), respectively), phenformin (lane 5, (A,B), respectively), or in glucose-free media for 8 h (lane 6, (A,B), respectively). Cells were also treated with a combination of salicylate (3 mM) and compound C (dorsomorphin 100 nM) for 48 h (lane 4, (A,B), respectively). In MCF-7 cells, the expression of c-Myc was significantly decreased by 4-OHT alone (lane 9) and even more by a combination of salicylate and 4-OHT (lane 10), compared with the negative control (lane 7). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in all cases as loading control for protein normalization. (C) Cell viability of HCT116 cells was determined by MTT assay after treatment with indicated concentrations of salicylate for 48 h. IC50 for salicylate was determined using Prism v.9 (GraphPad Software, San Diego, CA, USA) based on the changes in HCT116 cell viability. (D) Cell viability was determined after 48 h in the presence of 1 mM or 3 mM of salicylate, the synthetic activator A76966, compound C (dorsomorphin: 100 nM), or combinations of these drugs. Mean values ± SEMs are shown. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s.: not significant. (E,F) Seahorse extracellular flux analysis of the oxygen consumption rate (OCR) shows respiratory changes in HCT-116 cells brought about by sodium salicylate treatment. (E) Seahorse injection series analysis with OCR data presented for one experiment. (F) Calculated parameters from 3 independent experiments include ATP-linked respiration, proton leak, basal respiration, and maximal respiratory capacity. Basal respiration significantly increased when cells were treated with sodium salicylate (1 or 3 mM). Treatment with sodium salicylate caused an increased proton leak (uncoupling) and a decreased percental amount of oxygen being used for ATP production. The maximal respiratory capacity was lowered when treated with sodium salicylate, which suggests a compromised mitochondrial function.

Article Snippet: QPCR primer pairs for human MYC (#HP206146) and human GAPDH (#HP205798) were from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Negative Control, Control, MTT Assay, Software, Injection