acetyltransferase mutant  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: Inhibition of GCN5 activity results in a typical apoptosis. ( a ) DIV 7 CGNs treated with GCN5 inhibitors CPTH2, MB3, p300 inhibitor C646 or Tip60 inhibitor MG149 at the indicated doses for 12 h were subjected to WB to detect the level of Ac-H3K9, Ac-H3K27, Ac-H4K12, H3, H4 or GCN5. ( b and c ) CGNs treated with CPTH2 (50 μ M), MB-3 (150 μ M), C646 (10 μ M) or MG-149 (100 μ M) in 25K media for 24 h were subjected to nucleic staining or DNA ladder analysis. Fluorescent photos were taken on the Olympus IX71 microscope (scale bar=20 μ m). The white arrows indicate the apoptotic cells with nuclear pyknosis. The apoptotic rate was determined by scoring the percentage of cells with nuclear pyknosis in total Hoechst-stained ones. ( d and e ) CGNs treated with CPTH2, MB3 in 25K media at the indicated doses for 24 h and the active Caspase 3 by WB and apoptotic rate were determined as in panel ( b ). 5K treatment causes an active Caspase 3 as a positive control. ( f and g ) CGNs were treated with CPTH2 (50 μ M) or MB-3 (150 μ M) in 25K media for the indicated time courses, the active Caspase 3 by WB and apoptotic rate were determined as in panel ( b ). Data were presented as means±S.E.M., n =3, * P <0.05. DMSO, dimethyl sulphoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Inhibition, Activity Assay, Staining, Microscopy, Positive Control

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: Knockdown of GCN5 by siRNAs induces neuronal apoptosis. ( a ) Rat C6 glioma cells transfected with NC (non-targeted control siRNA), siGCN5a or siGCN5b were subjected to WB to detect the GCN5 expression. Tubulin was reprobed to verify equal loadings. ( b ) DIV5 CGNs transfected with siGCN5a and siGCN5b together with pCMV-Gal plasmids were subjected to immunofluorescence and photos were taken on confocal microscope (scale bar=5 μ m). β -Gal was stained to mark the transfected cells and the efficiency of RNA interference was determined by scoring the percentage of stained β -Gal-positive neuron population with GCN5. ( c ) CGNs transfected with siGCN5a and siGCN5b together with pCMV-EGFP plasmids were subjected to nucleic staining (scale bar=20 μ m), and the apoptotic rate was determined by scoring the percentage of GFP-positive neuron population with nuclear pyknosis. Mean±S.E.M., n =3, * P <0.05. GFP, green fluorescent protein

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Knockdown, Transfection, Control, Expressing, Immunofluorescence, Microscopy, Staining

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: Inhibition of GCN5 transcriptionally upregulates Bim. ( a ) CGNs treated with CPTH2 and MB-3 in 25K media at the indicated doses for 24 h and Bim was detected by WB. 5K treatment causes a Bim induction as a positive control. GAPDH was reprobed to verify equal loadings. ( b ) CGNs treated with CPTH2 (50 μ M) and MB-3 (150 μ M) in 25K media for the indicated time course were subjected to WB to detect Bim expression. ( c ) CGNs treated with CPTH2 (50 μ M) and MB-3 (150 μ M) in 25K media for 12 h were subjected to RT-PCR to detect bim mRNA level. The actin was amplified to verify equal loadings. ( d ) Pulse chase assay: CGNs treated with CPTH2 (50 μ M) in 25K media for 18 h were subjected to further co-treatment of CPTH2 with Actinomycin D (2 μ M) for 2, 4 and 8 h, and then to RT-PCR. Changes in bim mRNA levels were determined as changes in band intensity after normalization to actin by the ImageJ software. ( e ) DIV5 CGNs transfected with bim-luc reporter for 12 h were administered with CPTH2 (50 μ M) in 25K media for 24 h and subjected to dual reporter analysis. ( f ) CGNs transfected with NC, siGCN5a and siGCN5b were subjected to immunofluorescence to detect Bim expression (scale bar=10 μ m). ( g ) CGNs transfected with bim-luc reporter together with NC, siGCN5a and siGCN5b for 48 h were subjected to dual reporter analysis. Mean±S.E.M., n =3, * P <0.05. DMSO, dimethyl sulphoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Inhibition, Positive Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Pulse Chase, Software, Transfection, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: Knockdown of Bim rescues the loss of GCN5-induced apoptosis. ( a and b ) CGNs treated with CPTH2 at the indicated doses or 50 μ M in 25K for 24 h were subjected to WB or RT-PCR to detect Bcl-2, Mcl-1 or Bcl-XL expression. ( c ) Rat C6 glioma cells transfected with NC, siBima and siBimb were subjected to WB to detect Bim expression. ( d ) CGNs were transfected with NC, siBima and siBimb together with pCMV-Gal. Forty-eight hours later, neurons were exposed to CPTH2 (50 μ M) in 25K media for 24 h and subjected to immunofluorescence to detect Bim expression (scale bar=5 μ m). ( e ) CGNs were transfected with NC, siBima and siBimb together with pCMV-EGFP. Forty-eight hours later, neurons were exposed to CPTH2 (50 μ M) in 25K media for 24 h and subjected to nucleic staining (scale bar=20 μ m) and apoptotic analysis as in . White arrows indicate the transfected cells. ( f ) CGNs transfected with NC, siBima and siBimb together with siGCN5a and pCMV-EGFP plasmids were subjected to nucleic staining and apoptotic analysis as above. Mean±S.E.M., n =3, * P <0.05. DMSO, dimethyl sulphoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: GCN5 inhibition induces Bim expression through transactivating Egr-1 and E2F1. ( a ) CGNs were exposed to CPTH2 or dimethyl sulphoxide (DMSO) in 25K media for 18 h, small mRNAs were extracted and reverse transcribed as templates for Q-PCR to detect miR-24 expression and U6 small nuclear RNA (snRNA) was amplified to normalize the miR24 outputs. ( b ) CGNs in 25K media exposed to CPTH2 alone or together with indirubin-3'-oxime (10 μ M), flavopiridol (2 μ M), mithramycin A (10 μ M), chromomycin A3 (0.3 μ M), olomoucine (200 μ M), roscovitine (50 μ M), IGF (100 ng/ml), SP600125 (10 μ M), pifithrin-alpha (2 μ M) or JSH-23 (5 μ M) for 24 h were subjected to WB to detect Bim expression; tubulin was reprobed to verify equal loadings. ( c – e ) CGNs in 25K media treated with CPTH2 at the indicated doses for 24 h or treated with CPTH2 (50 μ M) for the indicated time courses were subjected to WB or RT-PCR to detect E2F1 and Egr-1 expression in protein or mRNA. ( f and g ) The cleaved chromatin fragments were immunoprecipitated by anti-E2F1 or Egr-1 antibody and then purified as templates to amplify the region of bim promoter by PCR. The part of chromatin fragments before IP was purified as templates to amplify bim promoter as a reference. ( h ) Rat C6 glioma cells transfected with NC, siE2F1 and siEgr-1 were subjected to WB to detect the E2F1 or Egr-1 expression. Tubulin was reprobed to verify equal loadings. ( i ) DIV5 CGNs transfected with NC, siE2F1 and siEgr-1 together with pCMV-Gal plasmids for 36 h were exposed to CPTH2 (50 μ M) in 25K media for 18 h and then were subjected to immunofluorescence to detect the Bim expression. The percentage of stained β -Gal-positive neuron population with Bim was calculated. ( j ) DIV5 CGNs transfected with NC, siE2F1 and siEgr-1 together with p-GFP plasmids for 36 h were exposed to CPTH2 (50 μ M) in 25K media for 24 h and then were subjected to nucleic staining and apoptotic analysis as in . Mean±S.E.M., n =3, * P <0.05

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Inhibition, Expressing, Reverse Transcription, Amplification, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Purification, Transfection, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: GCN5 loses its activity following potassium deprivation or glutamate exposure. ( a ) DIV 7 CGNs treated with 25K or 5K for 4 h, or glutamate (Glu, 100 μ M), were subjected to WB to detect Ac-H3K9, Ac-H3K14, Ac-H3K27, H3, Ac-H4K5, H4, Ac-H2BK5 and H2B. Con: control. ( b ) CGNs treated with 25K and 5K for two time durations 2 and 4 h were subjected to WB to detect Ac-H3K9, H3, E2F1, Egr-1 and Bim. ( c ) CGNs exposed to glutamate in 25K media for 8 h were subjected to WB to detect Ac-H3K9, H3, E2F1, Egr-1 and Bim. ( d and e ) CGNs treated with 25K and 5K for 4 h were subjected to IP with anti-GCN5 antibody and then to WB to detect the precipitated GCN5 (partial) and the left precipitated complexes were subjected to in vitro HAT activity. Mean±S.E.M., n =3, * P <0.05. GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Activity Assay, Control, In Vitro

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: Overexpression of GCN5 suppresses Bim expression and apoptosis following potassium deprivation or glutamate exposure. ( a ) DIV 5 CGNs were infected with Ad-GFP or Ad-GCN5 at 100 multiplicity of infection. After 48 h, the infected neurons were subjected to nucleic staining (scale bar=20 μ m) to determine infectious efficiency or RT-PCR to determine e2f1 , egr-1 and bim mRNA level. ( b and c ) Forty-eight hours posttransfection, the neurons infected as in panel ( a ) were subjected to 25K and 5K treatment for 6 h and then to RT-PCR to detect e2f1 , egr-1 , and bim mRNA level, and WB was performed to detect GCN5, Bim and Caspase 3. L.E.: long time of exposure; S.E.: short time of exposure. ( d ) Forty-eight hours posttransfection, the neurons infected as in panel ( a ) were subjected to 25K and 5K treatment for 12 h and the apoptotic rate was determined as in . ( e and f ) Forty-eight hours posttransfection, the neurons infected as in panel ( a ) were exposed to glutamate (Glu, 100 μ M) in 25K media for 12 h followed by RT-PCR to detect e2f1 , egr-1 and bim mRNA level or for 24 h and the apoptotic rate was determined as above. Mean±S.E.M., n =3, * P <0.05. GFP, green fluorescent protein

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Over Expression, Expressing, Infection, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: GCN5 loses its activity following SAH. ( a ) The location of GCN5 in the neurons of sham or SAH rat were determined by co-staining with GCN5 and NeuN, scale bar=20 μ m. ( b ) The protein expression levels or mRNA levels of GCN5 were determined by WB or RT-PCR at 24 h after SAH. ( c ) At 24 h after SAH, the homogenized brain tissues were subjected to a histone extraction followed by WB to detect Ac-H3K9, Ac-H3K14, Ac-H3K27, H3, Ac-H4K12, H4, Ac-H2BK5 and H2B. ( d and e ) At 24 h after SAH, the homogenized brain tissues were subjected to IP and the partial precipitated complexes were subjected to WB and the left ones were subjected to in vitro HAT activity assay. The values are expressed as mean±S.E.M., * P <0.05, n =6 in each group

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Activity Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Extraction, In Vitro, HAT Activity Assay

Journal: Cell Death & Disease

Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

doi: 10.1038/cddis.2016.465

Figure Lengend Snippet: pan-HDAC inhibitors suppress Bim expression and apoptosis induced by loss of GCN5, potassium deprivation or SAH. ( a and b ) CGNs in 25K media treated with DMSO, CPTH2 (50 μ M) or CPTH2 together with SAHA (1 μ M), TSA (0.5 μ M) and VPA (6 mM) for 24 h were subjected to WB to detect Bim and Caspase 3, Caspase 6 or subjected to nucleic staining to determine the apoptotic rates as in . ( c and d ) CGNs treated with 25K, 5K or 5K together with SAHA (1 μ M), TSA (0.5 μ M) and VPA (6 mM) for 12 h were subjected to WB to detect Bim and Caspase 3 or subjected to nucleic staining to determine the apoptotic rates. ( e and f ) The mRNA levels or protein expression levels of Bim, Egr-1, E2F1 and Bcl-2 were determined by RT-PCR or WB at 24 h after SAH in SAH+vehicle rats or SAH+SAHA ones. The activity of Caspase 3 was also determined. ( g and h ) At 24 h after SAH, TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labelling) or haematoxylin nd eosin (H&E) staining was performed to compare the difference of the apoptotic rate or the nuclear irregularity rate between the SAH+vehicle and SAH+SAHA groups. ( i and j ) At 24 h after SAH, the difference of neurobehavioral performance or brain water content in the left or right hemisphere (L.H or R.H.) was compared between the SAH+vehicle and SAH+SAHA groups. The values are expressed as mean±S.E.M., * P <0.05, n =6 in each group. ( k ) A schematic diagram illustrating the possible mechanisms involved in neuronal apoptosis following loss of GCN5 activity. In normal conditions, the activity of GCN5 is implicated in transcriptionally repressing Egr-1, E2F1 and Bim but promoting Bcl-2, and Mcl-1 expression to prevent Bax (or Bak) from permeabilizing the mitochondria for survival. When neurons are subjected to stress such as potassium deprivation or SAH, GCN5 loses its HAT activity, which leads to a HDAC activity-dependent upregulation of Egr-1 and E2F1 but a downregulation of Bcl-2 and Mcl-1(#no significant change between sham and SAH rats). The active Egr-1 or E2F1 transcriptionally upregulates Bim to activate Bax to increase the permeability of mitochondria for Cytochrome C and the reduction of Bcl-2 or Mcl-1 frees Bim proteins to facilitate the process. As a result, the Caspases are activated and apoptosis occurs. Inhibition of HDACs significantly rescues the stress-evoked events and neuronal apoptosis. DMSO, dimethyl sulphoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: The recombinant adenovirus plasmid AdEasy Flag GCN5 got from Addgene (plasmid no. 14106, Cambridge, MA, USA) was originally constructed by Dr Pere Puigserver's laboratory.

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Activity Assay, TUNEL Assay, Permeability, Inhibition