Journal: Nature Communications

Article Title: Hepatic p63 regulates steatosis via IKKβ/ER stress

doi: 10.1038/ncomms15111

Figure Lengend Snippet: ( a ) p53 protein levels in the liver after a tail vein injection of an associate adenovirus serotype 8 (AAV8) expressing either GFP or Cre in p53floxed mice fed a standard diet (STD) ( n =7 per group). Effects of the liver-specific silencing of p53 on ( b ) in hematoxylin-eosin (upper panel) and oil red O staining (lower panel) of mice liver sections ( n =3 per group); ( c ) total liver TG content, serum AST, ALT and TG levels ( n =8 AAV8-GFP and 10 AAV8-Cre mice); ( d ) liver protein levels of FAS, pJNK/JNK, pIRE/IRE, XBP1s, pPERK, peIF2α/eIF2α, cleaved caspase 3, cleaved caspase 7, ApoB100 and ApoB48; ( e ) mRNA expression of CPT1, ACADM, ACADL and FATP2; ( f ) serum ketone bodies; ( g ) glucose and ( h ) insulin tolerance test ( n =8 AAV8-GFP and 10 AAV8-Cre mice). The values of AAV8 GFP mice were always normalized to 100% ( n =7 per group). Protein GAPDH or transferrin levels were used to normalize protein levels. Dividing lines indicate splicings in the same gel. Uncropped blots of this Figure accompanied by the location of molecular weight markers are shown in . Data are presented as mean±standard error mean (s.e.m.). Statistical significance, * P <0.05 and ** P <0.01, was tested using Student t -test comparing AAV8-GFP and AAV8-Cre mice.

Article Snippet: Total protein lysates from liver (20 μg) and THLE2 cells (2 μg) were subjected to SDS-PAGE, electrotransferred onto polyvinylidene difluoride membranes (Millipore) and probed with the indicated antibodies: Fatty Acid Synthase (FAS) (H-300) (sc-20,140), JNK 1/3 (C-17) (sc-474), XBP-1 (M-186) (sc-7,160), p-PERK (TH981) (sc-32,577), p63 (H-129) (sc-11,386), elF2α (FL-315) (sc-11,386), pelF2α (Ser52) (sc-1,01,670), pIKKαβ (ser180/ser181) (sc-23,470-R), p21 (C19) (sc-397) (Santa Cruz Biotechnology, USA); Phospho-SAP/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (#4,668), Caspase 3 (#9,665), Cleaved Caspase 3 (Asp175) (#9,664), Caspase 7 (#9,492), Cleaved Caspase 7 (Asp 198) (#9,491), p53 (1C12) (#2,524), Phospho-p53-Ser15 Antibody (#9,284), Bax Antibody (#2,772) (Cell Signaling, USA); Anti-IKKβ [EPR6,043] (ab124,957), Anti-IKKβ (phosphor Y188) (ab1,94,519), Anti-p73 antibody (EP436Y) (ab40,658), Anti-IRE1 antibody (ab37,073), Anti-SHC (phosphor S36) [6E10] (ab54,518) (Abcam, UK); IRE 1 alpha [p ser 724] (NB100–2,323) (Novus Biologicals, USA); Anti-p63(TA) clone: Poly6,189 (cat:618,902), Anti-p63(ΔN) clone: Poly6,190 (cat:619,002) (Biolegend, USA); GFP (Living Colours , Clontech, Tokio, Japan).

Techniques: Injection, Expressing, Staining, Molecular Weight

Journal: Nature Communications

Article Title: Hepatic p63 regulates steatosis via IKKβ/ER stress

doi: 10.1038/ncomms15111

Figure Lengend Snippet: ( a ) GFP protein levels in the liver and brown adipose tissue (BAT) of WT and p53 null mice after a tail vein injection of adenoviruses encoding either GFP or p53 ( n =5 per group). ( b ) p53 gene expression in the liver of WT and p53 null mice after the injection of adenoviruses encoding either GFP or p53. ( c ) Representative photomicrographs of hematoxylin-eosin (upper panel) and oil red O staining (lower panel) of mice liver sections ( n =3 per group); ( d ) total liver TG content, serum AST and TG levels ( n =6 GFP and 5 p53 in WT mice; n =5 GFP and 4 p53 in KO mice); ( e ) mRNA expression of CPT1, ACADM, ACADL and FATP2; and ( f ) protein levels of FAS, pJNK/JNK, pIRE/IRE, XBP1s, pPERK, peIF2α/eIF2α, cleaved caspase 3, cleaved caspase 7, ApoB100 and ApoB48 in the liver of WT and p53 null mice fed a HFD after the over-expression of hepatic p53. Western blots were performed separately in WT and p53 null mice, and the values of Ad GFP mice were always set to 100% ( n =5 GFP and p53 in both WT and p53 null mice). GAPDH or transferrin were used to normalize protein levels. Dividing lines indicate splicings in the same gel. Uncropped blots of this Figure accompanied by the location of molecular weight markers are shown in . Data are presented as mean±standard error mean (s.e.m.). Statistical significance, * P <0.05 and ** P <0.01, was tested using Student t -test comparing WT and KO mice. ND: non detected.

Article Snippet: Total protein lysates from liver (20 μg) and THLE2 cells (2 μg) were subjected to SDS-PAGE, electrotransferred onto polyvinylidene difluoride membranes (Millipore) and probed with the indicated antibodies: Fatty Acid Synthase (FAS) (H-300) (sc-20,140), JNK 1/3 (C-17) (sc-474), XBP-1 (M-186) (sc-7,160), p-PERK (TH981) (sc-32,577), p63 (H-129) (sc-11,386), elF2α (FL-315) (sc-11,386), pelF2α (Ser52) (sc-1,01,670), pIKKαβ (ser180/ser181) (sc-23,470-R), p21 (C19) (sc-397) (Santa Cruz Biotechnology, USA); Phospho-SAP/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (#4,668), Caspase 3 (#9,665), Cleaved Caspase 3 (Asp175) (#9,664), Caspase 7 (#9,492), Cleaved Caspase 7 (Asp 198) (#9,491), p53 (1C12) (#2,524), Phospho-p53-Ser15 Antibody (#9,284), Bax Antibody (#2,772) (Cell Signaling, USA); Anti-IKKβ [EPR6,043] (ab124,957), Anti-IKKβ (phosphor Y188) (ab1,94,519), Anti-p73 antibody (EP436Y) (ab40,658), Anti-IRE1 antibody (ab37,073), Anti-SHC (phosphor S36) [6E10] (ab54,518) (Abcam, UK); IRE 1 alpha [p ser 724] (NB100–2,323) (Novus Biologicals, USA); Anti-p63(TA) clone: Poly6,189 (cat:618,902), Anti-p63(ΔN) clone: Poly6,190 (cat:619,002) (Biolegend, USA); GFP (Living Colours , Clontech, Tokio, Japan).

Techniques: Injection, Expressing, Staining, Over Expression, Western Blot, Molecular Weight

Journal: Nature Communications

Article Title: Hepatic p63 regulates steatosis via IKKβ/ER stress

doi: 10.1038/ncomms15111

Figure Lengend Snippet: ( a ) p63 protein levels in the liver of mice after a tail vein injection of an AAV8 over-expressing either GFP or TAp63α isoform. ( b ) Representative photomicrographs of hematoxylin-eosin (upper panel) and oil red O staining (lower panel) of mice liver sections ( n =4 per group); ( c ) total liver TG content, serum AST, ALT and TG levels ( n =9 AAV8-GFP and 10 AAV8-TAp63α); ( d ) protein levels of FAS, pJNK/JNK, pIRE/IRE, XBP1s, pPERK, peIF2α/eIF2α, cleaved caspase 3, cleaved caspase 7, ApoB100 and ApoB48; and ( e ) mRNA expression of CPT1, ACADM, ACADL and FATP2 in the liver of mice after hepatic over-expression of TAp63 and IP TUDCA administration ( n =7 per group). ( f ) GRP78 protein levels in the liver of mice after a tail vein injection of an Ad over-expressing either GFP or GRP78 ( n =6 per group). ( g ) Representative photomicrographs of hematoxylin-eosin (upper panel) and oil red O staining (lower panel) of mice liver sections ( n =4 per group); ( h ) total liver TG content, serum AST, TG and ketone bodies levels ( n =9 AAV8-GFP and 10 AAV8-TAp63α); ( i ) protein levels of FAS, pIRE/IRE, XBP1s, ApoB100 and ApoB48 ( n =6 per group); and ( j ) mRNA expression of CPT1, ACADM, ACADL and FATP2 in the liver of mice after hepatic over-expression of TAp63α and Ad GRP78 administration ( n =8 per group). Protein GAPDH or transferrin levels were used to normalize protein levels and control values (AAV8 GFP) were normalized to 100%. Dividing lines indicate splicings in the same gel ( n =7 per group). Uncropped blots of this Figure accompanied by the location of molecular weight markers are shown in and . Data are presented as mean±standard error mean (s.e.m.). Statistical significance, * P <0.05, ** P <0.01 and *** P <0.001. For multiple comparison ( c – e , h – j ) a one way ANOVA followed by Bonferroni or Kruskal-Wallis test was performed. Student t -test was used in TAp63alpha and GRP78 liver protein levels ( a , f ).

Article Snippet: Total protein lysates from liver (20 μg) and THLE2 cells (2 μg) were subjected to SDS-PAGE, electrotransferred onto polyvinylidene difluoride membranes (Millipore) and probed with the indicated antibodies: Fatty Acid Synthase (FAS) (H-300) (sc-20,140), JNK 1/3 (C-17) (sc-474), XBP-1 (M-186) (sc-7,160), p-PERK (TH981) (sc-32,577), p63 (H-129) (sc-11,386), elF2α (FL-315) (sc-11,386), pelF2α (Ser52) (sc-1,01,670), pIKKαβ (ser180/ser181) (sc-23,470-R), p21 (C19) (sc-397) (Santa Cruz Biotechnology, USA); Phospho-SAP/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (#4,668), Caspase 3 (#9,665), Cleaved Caspase 3 (Asp175) (#9,664), Caspase 7 (#9,492), Cleaved Caspase 7 (Asp 198) (#9,491), p53 (1C12) (#2,524), Phospho-p53-Ser15 Antibody (#9,284), Bax Antibody (#2,772) (Cell Signaling, USA); Anti-IKKβ [EPR6,043] (ab124,957), Anti-IKKβ (phosphor Y188) (ab1,94,519), Anti-p73 antibody (EP436Y) (ab40,658), Anti-IRE1 antibody (ab37,073), Anti-SHC (phosphor S36) [6E10] (ab54,518) (Abcam, UK); IRE 1 alpha [p ser 724] (NB100–2,323) (Novus Biologicals, USA); Anti-p63(TA) clone: Poly6,189 (cat:618,902), Anti-p63(ΔN) clone: Poly6,190 (cat:619,002) (Biolegend, USA); GFP (Living Colours , Clontech, Tokio, Japan).

Techniques: Injection, Expressing, Staining, Over Expression, Molecular Weight

Journal: Cell Death & Disease

Article Title: Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation

doi: 10.1038/cddis.2013.509

Figure Lengend Snippet: Antioxidants decrease the amount of 5-Fu-induced apoptosis in colon cancer cells. ( a ) HT29 and SW480 colon cancer cells were pretreated with NAC (10 mM, upper panel) or catalase (200 mU, lower panel) for 2 h and then 5-Fu (50 μ M) was added to the mixture and cells incubated for an additional 48 h. Apoptosis was determined by flow cytometry. Data are shown as means±S.D. of triplicate measurements. The asterisks indicate a significant decrease in apoptosis in cells treated with NAC or catalase (*** P <0.001). ( b ) HT29 and SW480 cells were treated as in a and harvested. Total and cleaved PARP, caspase-7 and caspase-3 were detected by western blot analysis using specific antibodies. Data shown are representative of results from triplicate independent experiments. ( c ) HT29 cells were pretreated with VC (50 μ g/ml) or GSH (40 μ M) for 2 h and then 5-Fu (50 μ M) was added to the mixture and cells were incubated for an additional 48 h. Apoptosis was measured by flow cytometry and data are shown as means±S.D. of triplicate measurements. The asterisks indicate a significant decrease in apoptosis in cells treated with VC or GSH (*** P <0.001)

Article Snippet: The antibody to detect total caspase-7 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Incubation, Flow Cytometry, Western Blot

Journal: Cell Death & Disease

Article Title: Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation

doi: 10.1038/cddis.2013.509

Figure Lengend Snippet: Src contributes to 5-Fu-induced apoptosis. ( a ) The protein level of Src in wild-type ( src +/+ ) and knockout ( src −/− ) MEFs was determined by western blotting. Sh-Mock and Src knockdown (sh-Src) cells were generated by stable infection with an sh-mock , sh-src#1 or sh-src#2 plasmid into HT29 and SW480 cells. Src protein levels were determined by western blotting. β -Actin was detected in the same membrane and served as a loading control. ( b ) Comparison of the cytotoxicity of 5-Fu against src +/+ and src −/− MEFs, or in sh-Mock- and sh-Src#1- or sh-Src#2-expressing cells. An MTS assay was used to assess cytotoxicity at 24 h for MEFs or 48 h for colon cancer cells after treatment with 5-Fu. Absorbance was read at 492 nm and data are expressed as percentage of untreated control (100%). Data are shown as means±S.D. of triplicate measurements. The asterisks indicate significantly (* P <0.05, ** P <0.01, *** P <0.001) less cytotoxicity induced by 5-Fu. ( c ) 5-Fu-induced apoptosis was determined by flow cytometry. Data are shown as means±S.D. of triplicate measurements. The asterisks indicate significantly less apoptosis induced by 5-Fu (** P <0.01, *** P <0.001). ( d ) 5-Fu induces less cleaved PARP, cleaved caspase-7 and cleaved caspase-3 in cells expressing low levels of Src. 5-Fu (50 μ M) was used to simulate MEFs and colon cancer cells. Total and cleaved PARP, caspase-7 and caspase-3 were detected by western blot analysis using specific antibodies. Data shown are representative of results from triplicate independent experiments. ( e ) Caspase-3/7 activity is decreased in cells expressing low levels of Src compared with cells expressing normal levels of Src after 5-Fu treatment. 5-Fu (50 μ M) was used to simulate cells. After 12 or 48 h, cells were harvested, and caspase-3/7 activity was detected as described in ‘Materials and Methods'. Data are shown as means±S.D. of triplicate measurements. The asterisks indicate significantly lower caspase 3/7 activity (** P <0.01, *** P <0.001)

Article Snippet: The antibody to detect total caspase-7 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Knock-Out, Western Blot, Generated, Infection, Plasmid Preparation, Expressing, MTS Assay, Flow Cytometry, Activity Assay

Journal: Cell Death & Disease

Article Title: Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation

doi: 10.1038/cddis.2013.509

Figure Lengend Snippet: Src phosphorylates caspase-7 in vitro and interacts with caspase-7 in cells. ( a ) Src phosphorylates caspase-7 in vitro . Results of an in vitro kinase assay in the presence of [ γ - 32 P] ATP were visualized by autoradiography. Coomassie Brilliant Blue staining was used to assure that the same quantity of protein was loaded. Data shown are representative of results from triplicate independent experiments. ( b ) Src phosphorylates caspase-7 at tyrosine residues as determined by western blotting following an in vitro kinase assay. IB, immunoblot. ( c ) Src binds with caspase-7 but not caspase-3 in colon cancer cells. HT29 and SW480 total cell lysates (1 mg) were immunoprecipitated with anti-caspase-3/7, and the immunoprecipitated complex was probed to detect Src. ( d ) Src and caspase-7 co-localize in SW480 cells. SW480 cells were seeded in four-chamber glass slides and treated or not treated with 5-Fu (50 μ M) for 48 h. Src and caspase-7 were detected by immunofluorescence staining with Src and caspase-7 primary antibodies, accompanied by the respective secondary antibody

Article Snippet: The antibody to detect total caspase-7 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: In Vitro, Kinase Assay, Autoradiography, Staining, Western Blot, Immunoprecipitation, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation

doi: 10.1038/cddis.2013.509

Figure Lengend Snippet: Src phosphorylates caspase-7 at multiple tyrosine sites. ( a ) Potential tyrosine phosphorylation sites of caspase-7 were predicted by the NetPhos 2.0 software program. ( b ) Peptide mapping of nine synthesized peptides containing potential tyrosine sites. Sites were examined using an in vitro kinase assay with Src in the presence of [ γ - 32 P] ATP and results were visualized by autoradiography. ( c ) Confirmation of the peptide mapping results using an in vitro kinase assay with Src and wild-type (Wt)-caspase-7 or mutant (Mut)-caspase-7. Results were visualized by autoradiography. Coomassie Brilliant Blue staining was used to assure that the same quantity of protein was loaded. For b and c , representative blots from three independent experiments are shown

Article Snippet: The antibody to detect total caspase-7 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Software, Synthesized, In Vitro, Kinase Assay, Autoradiography, Mutagenesis, Staining

Journal: Cell Death & Disease

Article Title: Antioxidants decrease the apoptotic effect of 5-Fu in colon cancer by regulating Src-dependent caspase-7 phosphorylation

doi: 10.1038/cddis.2013.509

Figure Lengend Snippet: Src enhances caspase-7 activity in vitro and in cells. ( a ) Phosphorylation of caspase-7 by Src increases caspase-7 activity. An in vitro kinase assay was performed using Src and a Wt-caspase-7 (0.8 μ g) or Mut-caspase-7 (0.8 μ g) protein. The reaction was then incubated with Wt-caspase-9 for 1 h at 32 °C and caspase-7 activity was detected at various time points as described in ‘Materials and Methods'. Data are shown as means±S.D. from triplicate experiments. The asterisks indicate a significant enhancement in caspase-7 activity (** P <0.01, *** P <0.001). ( b ) 293T cells were transiently transfected with HA-mock, Wt-myc-caspase-7 or Mut-myc-caspase-7 and at 24 h after transfection, cells were harvested. The level of Myc was detected by western blotting and caspase-7 activity was detected at various time points. Data are shown as means±S.D. from triplicate experiments. The asterisks indicate a significant enhancement in caspase-7 activity (** P <0.01, *** P <0.001). ( c ) 293T cells were transiently co-transfected with HA-mock/His-Src and Wt-myc-caspase-7/Mut-myc-caspase-7 and at 24 h after transfection, cells were harvested. His and Myc were detected by western blot using specific antibodies. Caspase-7 activity is shown as means±S.D. from triplicate experiments. The asterisks indicate a significant enhancement in caspase-7 activity (** P <0.01)

Article Snippet: The antibody to detect total caspase-7 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Activity Assay, In Vitro, Kinase Assay, Incubation, Transfection, Western Blot