Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) Co-IP of Sirt5 and TPI in CDCP1– and CDCP1+ fractions from mu Kras and wt Kras CRC cells. (B–C) DKs5 and HK2-10–derived CDCP1+ cells were treated with Au-H3K9Su for 48 h, after which lysine modifications (B) and enzyme activity (C) of endogenous TPI were determined. (D–E) DKs5 and HK2-10–derived CDCP1+ cells were transfected with Sirt5 shRNA or HA-H158Y mutant, after which lysine malonylation (D) and enzyme activity (E) of endogenous TPI were determined. (F–G) The indicated Flag-tagged TPI vectors were transfected into DKs5-derived CDCP1+ cells with stable Sirt5 KD or H158Y overexpression. WT and mutant TPI proteins were purified by Flag beads and eluted with Flag peptide, followed by determination of their lysine malonylation (F) and enzyme activity (G). All experiments were independently repeated three times in triplicate. Values shown are mean ± SD. A two-tailed unpaired t test was used to compare experimental groups. ** p < 0.05. Underlying data are available in . acetyl-K, lysine acetylation; CD, cluster of differentiation; CDCP1, CUB-domain–containing protein 1; CRC, colorectal carcinoma; glu-K, lysine glutarylation; HA, hemagglutinin; IP, immunoprecipitation; KD, knockdown; Kras , Kirsten rat sarcoma viral oncogene homolog; mal-K, lysine malonylation; mu Kras , mutant Kras ; shRNA, short hairpin RNA; Sirt5, silent mating type information regulation 2 homolog 5; succ-K, lysine succinylation; TPI, triosephosphate isomerase; WB, western blot; WT, wild type; wt Kras , wild-type Kras .

Article Snippet: The primary antibodies used for immunohistochemistry, immunofluorescence, immunoprecipitation, and western blotting analysis were as follows: CDCP1 (Cell Signaling Technology #4115; Danvers, MA, USA), CDCP1 (Abcam #188818; Cambridge, UK), IdU (Abcam #181664), BrdU (Abcam #6326), HA (Abcam #18181), myc (Abcam #32072), Kras (Santa Cruz Biotechnology #sc-30; Dallas, TX, USA), Flag (Sigma-Aldrich #F1084), malonyl-lysine (PTM Biolabs 901; Chicago, IL, USA), succinyl-lysine (PTM Biolabs 401), glutaryl-lysine (PTM Biolabs 1151), acetyl-lysine (PTM Biolab 104), Sirt5 (Cell Signaling Technology 8782), tubulin (Abcam 7291), and TPI (Abcam #96696).

Techniques: Co-Immunoprecipitation Assay, Derivative Assay, Activity Assay, Transfection, shRNA, Mutagenesis, Over Expression, Purification, Two Tailed Test, Immunoprecipitation, Knockdown, Western Blot

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) Malonylation of K56 (red) induces the formation of hydrogen bonds with D88 (pink) and Q91 (blue), stabilizing the dimerization of TPI. (B) Close-up view of the interactions at the TPI dimer interface with K56 (Protein Data Bank: 4POC). Four out of the eight contacts dependent on K56 malonylation are indicated with a dashed line. The other four hydrogen bonds are the equivalent companions on the reverse side of the protein and are not visible in the picture. (C) Flag-tagged TPI or K56A/K56E mutants were each expressed in HK2-10–derived CDCP1+ cells coexpressing HA-tagged TPI. The interaction between Flag-tagged and HA-tagged proteins was determined by western blot. (D) Flag-tagged TPI or K56A/K56E mutants were each expressed in HEK293T cells, followed by treatments with or without 0.025% glutaraldehyde. The formation of TPI monomer and dimer was determined by western blot. (E) Flag-tagged TPI or K56E mutant was each expressed in HEK293T cells coexpressing HA-tagged TPI or K56E mutant, respectively. Cells were then transfected with or without myc-Sirt5, and the interaction between Flag-tagged and HA-tagged proteins was determined by western blot. CDCP1, CUB-domain–containing protein 1; HA, hemagglutinin; HEK, human embryonic kidney; IP, immunoprecipitation; MW, molecular weight; Sirt5, silent mating type information regulation 2 homolog 5; TPI, triosephosphate isomerase; WB, western blot; WT, wild type.

Article Snippet: The primary antibodies used for immunohistochemistry, immunofluorescence, immunoprecipitation, and western blotting analysis were as follows: CDCP1 (Cell Signaling Technology #4115; Danvers, MA, USA), CDCP1 (Abcam #188818; Cambridge, UK), IdU (Abcam #181664), BrdU (Abcam #6326), HA (Abcam #18181), myc (Abcam #32072), Kras (Santa Cruz Biotechnology #sc-30; Dallas, TX, USA), Flag (Sigma-Aldrich #F1084), malonyl-lysine (PTM Biolabs 901; Chicago, IL, USA), succinyl-lysine (PTM Biolabs 401), glutaryl-lysine (PTM Biolabs 1151), acetyl-lysine (PTM Biolab 104), Sirt5 (Cell Signaling Technology 8782), tubulin (Abcam 7291), and TPI (Abcam #96696).

Techniques: Derivative Assay, Western Blot, Mutagenesis, Transfection, Immunoprecipitation, Molecular Weight

Journal: PLoS Biology

Article Title: Targeting oxidative pentose phosphate pathway prevents recurrence in mutant Kras colorectal carcinomas

doi: 10.1371/journal.pbio.3000425

Figure Lengend Snippet: (A) Western blot analysis of CDCP1 cleavage and plasmin secretion in mu Kras and wt Kras CRCs. (B) Western blot analysis of CDCP1 cleavage in HKe3-derived CDCP1+ cells overexpressing Flag plasminogen. (C) HKe3-10–derived CDCP1+ cells were transfected with the Flag-plasminogen vector, together with or without 5-FU (5 μM) or oxaliplatin (1 μM) treatment. The apoptotic rate was measured at 48 h after drug treatment. (D) CRC108-derived CDCP1+ cells were transfected with siRNA targeting Kras , together with or without the HA-AktCA or Flag-MEKCA mutant, following which CDCP1 cleavage and plasmin expression were analyzed by western blot. (E) Analysis of CDCP1 cleavage in CRC108-derived CDCP1+ cells pretreated with 20 μg/ml CDCP1 mAb 10-D7 or control IgG for 24 h. (F) CRC108-derived CDCP1+ cells were pretreated with 20 μg/ml 10-D7 and/or HA-PKCδCA mutant for 48 h prior to assessment of Sirt5–TPI association and TPI K56 malonylation. (G) cCDCP1 expression in CRC108-derived CDCP1– cells overexpressing Flag-cCDCP1. (H) CRC108-derived CDCP1− cells transfected with Flag-cCDCP1 were treated with or without rottlerin (2.5 μM) for 48 h prior to assessment of Sirt5–TPI association and TPI K56 malonylation. Underlying data are available in . Akt, RAC-alpha serine/threonine-protein kinase; AktCA, catalytically active mutant of Akt; cCDCP1, cleaved CDCP1; CDCP1, CUB-domain–containing protein 1; CRC, colorectal carcinoma; fCDCP1, full-length CDCP1; HA, hemagglutinin; IgG, immunoglobulin G; IP, immunoprecipitation; Kras , Kirsten rat sarcoma viral oncogene homolog; mAb, monoclonal antibody; malTPI, malonylated TPI; MEK, mitogen-activated protein kinase; MEKCA, catalytically active mutant of MEK; mu Kras , mutant Kras ; MW, molecular weight; PKCδ, protein kinase C-delta; PKCδCA, catalytically active mutant of PKCδ; Si-KRAS, KRAS siRNA; siRNA, small interfering RNA; Sirt5, silent mating type information regulation 2 homolog 5; TPI, triosephosphate isomerase; WB, western blot; wt Kras , wild-type Kras ; 5-FU, 5-fluorpyrimidine.

Article Snippet: The primary antibodies used for immunohistochemistry, immunofluorescence, immunoprecipitation, and western blotting analysis were as follows: CDCP1 (Cell Signaling Technology #4115; Danvers, MA, USA), CDCP1 (Abcam #188818; Cambridge, UK), IdU (Abcam #181664), BrdU (Abcam #6326), HA (Abcam #18181), myc (Abcam #32072), Kras (Santa Cruz Biotechnology #sc-30; Dallas, TX, USA), Flag (Sigma-Aldrich #F1084), malonyl-lysine (PTM Biolabs 901; Chicago, IL, USA), succinyl-lysine (PTM Biolabs 401), glutaryl-lysine (PTM Biolabs 1151), acetyl-lysine (PTM Biolab 104), Sirt5 (Cell Signaling Technology 8782), tubulin (Abcam 7291), and TPI (Abcam #96696).

Techniques: Western Blot, Derivative Assay, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Control, Immunoprecipitation, Molecular Weight, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Abnormal expression of SIRTs in psoriasis: Decreased expression of SIRT 1-5 and increased expression of SIRT 6 and 7

doi: 10.3892/ijmm.2019.4173

Figure Lengend Snippet: Primers used for human reverse transcription-quantitative polymerase chain reaction in the present study.

Article Snippet: Cultured HaCaT cells were incubated with the following specific antibodies overnight at 4°C: SIRT1 (1:50; cat. no. 13161-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), SIRT2 (1:100; cat. no. PB0174; Wuhan Boster Biological Technology, Ltd., Wuhan, China), SIRT3 (1:100; cat. no. A01061-1; Wuhan Boster Biological Technology, Ltd.), SIRT4 (1:100; cat. no. ab10140; Abcam, Cambridge, MA, USA), SIRT5 (1:100; cat. no. 15122-1-AP; ProteinTech Group, Inc.), SIRT6 (1:100; cat. no. PB0375; Wuhan Boster Biological Technology, Ltd.) and SIRT7 (1:100 cat. no. PB0376; Wuhan Boster Biological Technology, Ltd.).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Abnormal expression of SIRTs in psoriasis: Decreased expression of SIRT 1-5 and increased expression of SIRT 6 and 7

doi: 10.3892/ijmm.2019.4173

Figure Lengend Snippet: Primers used for mouse reverse transcription-quantitative polymerase chain reaction in the present study.

Article Snippet: Cultured HaCaT cells were incubated with the following specific antibodies overnight at 4°C: SIRT1 (1:50; cat. no. 13161-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), SIRT2 (1:100; cat. no. PB0174; Wuhan Boster Biological Technology, Ltd., Wuhan, China), SIRT3 (1:100; cat. no. A01061-1; Wuhan Boster Biological Technology, Ltd.), SIRT4 (1:100; cat. no. ab10140; Abcam, Cambridge, MA, USA), SIRT5 (1:100; cat. no. 15122-1-AP; ProteinTech Group, Inc.), SIRT6 (1:100; cat. no. PB0375; Wuhan Boster Biological Technology, Ltd.) and SIRT7 (1:100 cat. no. PB0376; Wuhan Boster Biological Technology, Ltd.).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Abnormal expression of SIRTs in psoriasis: Decreased expression of SIRT 1-5 and increased expression of SIRT 6 and 7

doi: 10.3892/ijmm.2019.4173

Figure Lengend Snippet: mRNA expression levels of SIRTs determined by reverse transcription-quantitative polymerase chain reaction in (A) cells, (B) mouse tissue and (C) human tissue. The expression levels of SIRT1, SIRT2, SIRT3, SIRT4 and SIRT5 in the experimental groups/patients with psoriasis were significantly reduced and those of SIRT6 and SIRT7 were significantly increased compared with the control groups. Error bars present the standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 vs. the control group. SIRT, Sirtuin.

Article Snippet: Cultured HaCaT cells were incubated with the following specific antibodies overnight at 4°C: SIRT1 (1:50; cat. no. 13161-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), SIRT2 (1:100; cat. no. PB0174; Wuhan Boster Biological Technology, Ltd., Wuhan, China), SIRT3 (1:100; cat. no. A01061-1; Wuhan Boster Biological Technology, Ltd.), SIRT4 (1:100; cat. no. ab10140; Abcam, Cambridge, MA, USA), SIRT5 (1:100; cat. no. 15122-1-AP; ProteinTech Group, Inc.), SIRT6 (1:100; cat. no. PB0375; Wuhan Boster Biological Technology, Ltd.) and SIRT7 (1:100 cat. no. PB0376; Wuhan Boster Biological Technology, Ltd.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Abnormal expression of SIRTs in psoriasis: Decreased expression of SIRT 1-5 and increased expression of SIRT 6 and 7

doi: 10.3892/ijmm.2019.4173

Figure Lengend Snippet: Protein expression levels of SIRTs determined by western blotting in (A) cells, (B) mouse tissue and (C) human tissue. The protein expression levels of SIRT1, SIRT2, SIRT3, SIRT4 and SIRT5 in the experimental groups/patients with psoriasis were significantly reduced and those of SIRT6 and SIRT7 were significantly increased compared with the controls. Error bars present the standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 vs. the control group. SIRT, Sirtuin.

Article Snippet: Cultured HaCaT cells were incubated with the following specific antibodies overnight at 4°C: SIRT1 (1:50; cat. no. 13161-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), SIRT2 (1:100; cat. no. PB0174; Wuhan Boster Biological Technology, Ltd., Wuhan, China), SIRT3 (1:100; cat. no. A01061-1; Wuhan Boster Biological Technology, Ltd.), SIRT4 (1:100; cat. no. ab10140; Abcam, Cambridge, MA, USA), SIRT5 (1:100; cat. no. 15122-1-AP; ProteinTech Group, Inc.), SIRT6 (1:100; cat. no. PB0375; Wuhan Boster Biological Technology, Ltd.) and SIRT7 (1:100 cat. no. PB0376; Wuhan Boster Biological Technology, Ltd.).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: International Journal of Molecular Medicine

Article Title: Abnormal expression of SIRTs in psoriasis: Decreased expression of SIRT 1-5 and increased expression of SIRT 6 and 7

doi: 10.3892/ijmm.2019.4173

Figure Lengend Snippet: IF in human skin lesions. IF in human psoriasis skin lesions was negative for (A) SIRT1, (B) SIRT2, (C) SIRT3, (D) SIRT4 and (E) SIRT5 antibodies. Lesions were positive for (F) SIRT6 and (G) SIRT7 antibodies. SIRTs were mainly observed in the epithelial layer. IF, immunofluorescence; SIRT, Sirtuin; FITC, fluorescein isothiocyanate.

Article Snippet: Cultured HaCaT cells were incubated with the following specific antibodies overnight at 4°C: SIRT1 (1:50; cat. no. 13161-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), SIRT2 (1:100; cat. no. PB0174; Wuhan Boster Biological Technology, Ltd., Wuhan, China), SIRT3 (1:100; cat. no. A01061-1; Wuhan Boster Biological Technology, Ltd.), SIRT4 (1:100; cat. no. ab10140; Abcam, Cambridge, MA, USA), SIRT5 (1:100; cat. no. 15122-1-AP; ProteinTech Group, Inc.), SIRT6 (1:100; cat. no. PB0375; Wuhan Boster Biological Technology, Ltd.) and SIRT7 (1:100 cat. no. PB0376; Wuhan Boster Biological Technology, Ltd.).

Techniques: Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: Abnormal expression of SIRTs in psoriasis: Decreased expression of SIRT 1-5 and increased expression of SIRT 6 and 7

doi: 10.3892/ijmm.2019.4173

Figure Lengend Snippet: IF in mouse skin lesions. IF in mouse skin lesions were also negative for (A) SIRT1, (B) SIRT2, (C) SIRT3, (D) SIRT4 and (E) SIRT5 antibodies. Lesions were positive for (F) SIRT6 and (G) SIRT7 antibodies. SIRTs were mainly localized in the epithelial layer. IF, immunofluorescence; SIRT, Sirtuin; FITC, fluorescein isothiocyanate.

Article Snippet: Cultured HaCaT cells were incubated with the following specific antibodies overnight at 4°C: SIRT1 (1:50; cat. no. 13161-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), SIRT2 (1:100; cat. no. PB0174; Wuhan Boster Biological Technology, Ltd., Wuhan, China), SIRT3 (1:100; cat. no. A01061-1; Wuhan Boster Biological Technology, Ltd.), SIRT4 (1:100; cat. no. ab10140; Abcam, Cambridge, MA, USA), SIRT5 (1:100; cat. no. 15122-1-AP; ProteinTech Group, Inc.), SIRT6 (1:100; cat. no. PB0375; Wuhan Boster Biological Technology, Ltd.) and SIRT7 (1:100 cat. no. PB0376; Wuhan Boster Biological Technology, Ltd.).

Techniques: Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: Abnormal expression of SIRTs in psoriasis: Decreased expression of SIRT 1-5 and increased expression of SIRT 6 and 7

doi: 10.3892/ijmm.2019.4173

Figure Lengend Snippet: IF in TNF-α-stimulated HaCaT cells was negative except for SIRT6 and 7. (A) SIRT1 and (B) SIRT2 were located predominantly in the nucleus and cytoplasm. (C) SIRT3, (D) SIRT4 and (E) SIRT5 were primarily mitochondrial proteins. (F) SIRT6 and (G) SIRT7 were mainly nuclear Sirtuins. IF, immunofluorescence; SIRT, Sirtuin; FITC, fluorescein isothiocyanate.

Article Snippet: Cultured HaCaT cells were incubated with the following specific antibodies overnight at 4°C: SIRT1 (1:50; cat. no. 13161-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), SIRT2 (1:100; cat. no. PB0174; Wuhan Boster Biological Technology, Ltd., Wuhan, China), SIRT3 (1:100; cat. no. A01061-1; Wuhan Boster Biological Technology, Ltd.), SIRT4 (1:100; cat. no. ab10140; Abcam, Cambridge, MA, USA), SIRT5 (1:100; cat. no. 15122-1-AP; ProteinTech Group, Inc.), SIRT6 (1:100; cat. no. PB0375; Wuhan Boster Biological Technology, Ltd.) and SIRT7 (1:100 cat. no. PB0376; Wuhan Boster Biological Technology, Ltd.).

Techniques: Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Sirtuin 5 regulates the proliferation, invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

doi: 10.1111/jcmm.16016

Figure Lengend Snippet: SIRT5 is highly expressed in prostate cancer tissues (A) Expression of SIRT5 protein in prostate tissue sections. (a) Normal prostate gland. (b) Prostate cancer tissue with Gleason score of 3. (c) Prostate cancer tissue with Gleason score of 4. (d) Prostate cancer tissue with Gleason score of 5. (B) Mutation type and rate of the SIRT5 gene in prostate cancer. (C) Mutation sites of the SIRT5 gene in prostate cancer; it is a L301I mutation. (D) Effect of SIRT5 gene mutations on the prognosis of prostate cancer ( P = .771). (E) Expression of SIRT5 in representative cell lines of a variety of tumours. SIRT5 expression is high in PC‐3 cells, a representative prostate cancer cell line. (F) Same as E, expression of SIRT5 in representative cell lines of a variety of tumours. SIRT5 expression is high in PC‐3 cells, a representative prostate cancer cell line. Data were obtained from Shankavaram CellLine (Oncomine)

Article Snippet: Cells were treated with the following: SIRT5 inhibitor 1 (HY‐112634, MCE, New Jersey, USA), a specific inhibitor of SIRT5, at a concentration of 0.1 μmol\L for 6 hours; K‐604 dihydrochloride (HY‐100400A, MCE), a specific inhibitor of ACAT1, at a concentration of 0.4 μmol\L for 6 hours; or MG‐132 (HY‐13259, MCE), a proteasome inhibitor that can effectively prevent hydrolysis of the 26S proteasome, at a concentration of 18.5 μmol\L for 24 hours.

Techniques: Expressing, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: Sirtuin 5 regulates the proliferation, invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

doi: 10.1111/jcmm.16016

Figure Lengend Snippet: Expression of SIRT5 in prostate cancer tissues

Article Snippet: Cells were treated with the following: SIRT5 inhibitor 1 (HY‐112634, MCE, New Jersey, USA), a specific inhibitor of SIRT5, at a concentration of 0.1 μmol\L for 6 hours; K‐604 dihydrochloride (HY‐100400A, MCE), a specific inhibitor of ACAT1, at a concentration of 0.4 μmol\L for 6 hours; or MG‐132 (HY‐13259, MCE), a proteasome inhibitor that can effectively prevent hydrolysis of the 26S proteasome, at a concentration of 18.5 μmol\L for 24 hours.

Techniques: Expressing, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Sirtuin 5 regulates the proliferation, invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

doi: 10.1111/jcmm.16016

Figure Lengend Snippet: SIRT5 promotes cell proliferation and migration (A) MTS cell proliferation experiments. The effect of SIRT5 silencing or high expression on the proliferation of LNCaP (left) and PC‐3 (right) cells. * P < .05, ** P < .01. (B) Colony formation experiments. After altering the expression of SIRT5, the colony forming ability of LNCaP cells and PC‐3 cells changed. The right panel shows a comparison of the number of colonies generated by these two cell lines. * P < .05, ** P < .01. (C) Transwell migration experiment. After altering the expression of SIRT5, the numbers of migrating LNCaP and PC‐3 cells changed. The right panel shows a comparison of the number of migrating cells of these two cell lines. * P < .05, ** P < .01. (D) High expression of SIRT5 in PC‐3 cells and LNCaP cells has the function of scavenging reactive oxygen species (ROS). * P < .05, ** P < .01

Article Snippet: Cells were treated with the following: SIRT5 inhibitor 1 (HY‐112634, MCE, New Jersey, USA), a specific inhibitor of SIRT5, at a concentration of 0.1 μmol\L for 6 hours; K‐604 dihydrochloride (HY‐100400A, MCE), a specific inhibitor of ACAT1, at a concentration of 0.4 μmol\L for 6 hours; or MG‐132 (HY‐13259, MCE), a proteasome inhibitor that can effectively prevent hydrolysis of the 26S proteasome, at a concentration of 18.5 μmol\L for 24 hours.

Techniques: Migration, Expressing, Comparison, Generated

Journal: Journal of Cellular and Molecular Medicine

Article Title: Sirtuin 5 regulates the proliferation, invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

doi: 10.1111/jcmm.16016

Figure Lengend Snippet: SIRT5 regulates the expression of mitogen‐activated protein kinase (MAPK) signalling proteins (A), (B) Effect of SIRT5 protein changes on the expression of functional and pathway proteins in (A) LNCaP and (B) PC‐3 cells. * P < .05, ** P < .01

Article Snippet: Cells were treated with the following: SIRT5 inhibitor 1 (HY‐112634, MCE, New Jersey, USA), a specific inhibitor of SIRT5, at a concentration of 0.1 μmol\L for 6 hours; K‐604 dihydrochloride (HY‐100400A, MCE), a specific inhibitor of ACAT1, at a concentration of 0.4 μmol\L for 6 hours; or MG‐132 (HY‐13259, MCE), a proteasome inhibitor that can effectively prevent hydrolysis of the 26S proteasome, at a concentration of 18.5 μmol\L for 24 hours.

Techniques: Expressing, Functional Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Sirtuin 5 regulates the proliferation, invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

doi: 10.1111/jcmm.16016

Figure Lengend Snippet: SIRT5 can combine with acetyl‐CoA acetyltransferase 1 (ACAT1) and regulate its expression (A) Mass spectrometry analysis on LNCaP cells showing that the ACAT1 protein can interact with the SIRT5 protein. (B) CAT1 mRNA levels in LNCaP and PC‐3 cells are unchanged irrespective of the SIRT5 content. * P < .05, ** P < .01. (C) When the expression of SIRT5 protein is altered in (a) LNCaP and (c) PC‐3 cells, the expression of ACAT1 protein also changes. * P < .05, ** P < .01. When the expression of ACAT1 protein is altered in (b) LNCaP and (d) PC‐3 cells, the expression of SIRT5 protein is unchanged. (D) Quantitative co‐precipitation experiments of SIRT5 and ACAT1 proteins. (a) Transfection of SIRT5 increases its expression in LNCaP cells. (b) Transfection of ACAT1 increases its expression in LNCaP cells and co‐immunoprecipitation experiments reveal that the expression of SIRT5 protein is also increased. (c) Transfection of SIRT5 increases its expression in PC‐3 cells and co‐immunoprecipitation experiments reveal that the expression of ACAT1 protein is also increased. (d) Transfection of ACAT1 increases its expression in PC‐3 cells and co‐immunoprecipitation experiments reveal that the expression of SIRT5 protein is also increased. (E) Confocal experiments show that SIRT5 and ACAT1 proteins co‐localize in LNCaP and PC‐3 cells

Article Snippet: Cells were treated with the following: SIRT5 inhibitor 1 (HY‐112634, MCE, New Jersey, USA), a specific inhibitor of SIRT5, at a concentration of 0.1 μmol\L for 6 hours; K‐604 dihydrochloride (HY‐100400A, MCE), a specific inhibitor of ACAT1, at a concentration of 0.4 μmol\L for 6 hours; or MG‐132 (HY‐13259, MCE), a proteasome inhibitor that can effectively prevent hydrolysis of the 26S proteasome, at a concentration of 18.5 μmol\L for 24 hours.

Techniques: Expressing, Mass Spectrometry, Transfection, Immunoprecipitation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Sirtuin 5 regulates the proliferation, invasion and migration of prostate cancer cells through acetyl‐CoA acetyltransferase 1

doi: 10.1111/jcmm.16016

Figure Lengend Snippet: SIRT5 regulates the function of prostate cancer cells through ACAT1 Overexpression of SIRT5 and silencing of ACAT1 in (A) LNCaP cells and (B) PC‐3 cells were performed simultaneously. Changes in the expression of other proteins in these two cell lines are shown. * P < .05, ** P < .01. (C) Changes of LNCaP (upper panel) and PC‐3 (lower panel) cell proliferation (MTS assay) after co‐transfection with the SIRT5 plasmid and ACAT1 siRNA. The results show that silencing ACAT1 prevents the SIRT5 tumour‐promotion effect. * P < .05, ** P < .01. (D) The colony formation assay shows that the ability of SIRT5 to promote colony formation is weakened in both LNCaP and PC‐3 cells after co‐transfection of the SIRT5 plasmid and ACAT1 siRNA. The right panel shows the number of colonies generated by these two cell lines. * P < .05,** P < .01. (E) The Transwell migration assay shows that the ability of SIRT5 to promote cell migration is weakened after co‐transfection of the SIRT5 plasmid and ACAT1 siRNA in both LNCaP and PC‐3 cells. * P < .05,** P < .01

Article Snippet: Cells were treated with the following: SIRT5 inhibitor 1 (HY‐112634, MCE, New Jersey, USA), a specific inhibitor of SIRT5, at a concentration of 0.1 μmol\L for 6 hours; K‐604 dihydrochloride (HY‐100400A, MCE), a specific inhibitor of ACAT1, at a concentration of 0.4 μmol\L for 6 hours; or MG‐132 (HY‐13259, MCE), a proteasome inhibitor that can effectively prevent hydrolysis of the 26S proteasome, at a concentration of 18.5 μmol\L for 24 hours.

Techniques: Over Expression, Expressing, MTS Assay, Cotransfection, Plasmid Preparation, Colony Assay, Generated, Transwell Migration Assay, Migration

Journal: PLoS ONE

Article Title: Age-Related Decrease in the Mitochondrial Sirtuin Deacetylase Sirt3 Expression Associated with ROS Accumulation in the Auditory Cortex of the Mimetic Aging Rat Model

doi: 10.1371/journal.pone.0088019

Figure Lengend Snippet: A. Endogenous SOD2 was immunopurified from the auditory cortex with anti-SOD2 antibody, followed by western blotting with anti-SIRT3, anti-Sirt4 and anti-Sirt5 antibodies. B. Endogenous Sirt3 was immunopurified from the auditory cortex with anti-Sirt3 antibody, followed by western blotting with anti-SOD2, anti-SOD1 and anti-SOD3 antibodies.

Article Snippet: The supernatant was then collected by centrifugation and subjected to western blotting analyses with anti-acetyl-lysine antibody (1∶1000, Cell Signaling Technology), anti-SOD1(1∶500, Santa Cruz), anti-SOD2 (1∶500, Santa Cruz), anti-SOD3 (1∶500, Santa Cruz), anti-Sirt3 (1∶500, Santa Cruz), anti-Sirt4 (1∶500, Santa Cruz), and anti-Sirt5 antibodies (1∶500, Santa Cruz).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

doi: 10.1083/jcb.201404118

Figure Lengend Snippet: Human ATP synthase β is an acetylated protein, and its deacetylation is regulated by SIRT3. (A) pCMV vector or ATP synthase β (DDK tagged) was transfected in HEK293T cells, immunoprecipitated using an antibody to DDK tag, and probed with an antibody to acetyl-Lys (Ac-Lys). (B) HEK293T cells were cotransfected with ATP synthase β (ATP syn β) and either SIRT3 siRNA or scrambled siRNA. ATP synthase β was immunoprecipitated, and its acetylation status was assessed. The bottom blot shows reduction of SIRT3 protein upon siRNA treatment. Knockdown of SIRT3 increases acetylation of ATP synthase β. (C) Expression vector for wild-type SIRT3 was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. Overexpression of SIRT3 decreases acetylation of ATP synthase β. (D) HEK293T cells were cotransfected with ATP synthase β and either SIRT4 siRNA or scrambled siRNA. SIRT4 knockdown does not affect acetylation of ATP synthase β. (E) Wild-type SIRT4 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT4 overexpression does not affect acetylation of ATP synthase β. (F) HEK293T cells were cotransfected with ATP synthase β and either SIRT5 siRNA or scrambled siRNA. SIRT5 knockdown does not affect acetylation of ATP synthase β. (G) Wild-type SIRT5 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT5 overexpression does not affect acetylation of ATP synthase β. (H) HEK293T cells were cotransfected with ATP synthase β and either SIRT1 siRNA or scrambled siRNA. SIRT1 knockdown does not affect acetylation of ATP synthase β. (I) Wild-type SIRT1 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT1 overexpression does not affect acetylation of ATP synthase β. (J) Mitochondria were prepared from SIRT3 siRNA–treated or scrambled siRNA–treated cells, and complex V activity was measured. The activity of mitochondria from scrambled siRNA treatment was taken as 100%. SIRT3 knockdown results in an ∼40% decrease in complex V activity. n = 3; error bars represent SDs. **, P ≤ 0.01–0.001 in Student’s t test. (K) Endogenous ATP synthase β was immunoprecipitated from HEK293T cells overexpressing SIRT3, and the immunoprecipitate was probed with antibodies to ATP synthase β and SIRT3. SIRT3 can coimmunoprecipitate with ATP synthase β. IP, immunoprecipitation; WB, Western blot.

Article Snippet: For siRNA experiments, cells were transfected with each siRNA (1 μM) or the scrambled version, and cells were harvested after 72 h. The Trilencer siRNAs used to reduce SIRT3 (SR308255), SIRT4 (SR308254), SIRT5 (SR308253), SIRT1 (SR308256), and the scrambled siRNAs were obtained from OriGene.

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Knockdown, Expressing, Over Expression, Activity Assay, Western Blot

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rb1 attenuates coronary microvascular inflammatory injury via NDUFS4-SIRT5-DUSP1-mediated mitochondrial quality control in a murine ischemia-reperfusion model

doi: 10.1016/j.jgr.2025.04.006

Figure Lengend Snippet: Ginsenoside Rb1 Ameliorates Excessive Inflammatory Response and Single-Cell Transcriptome Sequencing in I/R-Injured Myocardium. (A) HE staining to observe myocardial histopathological changes before and after I/R drug intervention (B–E) Detection of LDH, Troponin T, BNP, and CK-MB levels in each group to assess the severity of myocardial injury (F–H) Elisa to detect IL-10/IL-17/IL-1β expression levels in each group to assess the degree of inflammatory response (I) Immunofluorescence detection of Gr 1 and Tnt expression to determine the severity of myocardial injury (J–P) RT-PCR was used to detect the transcript levels of MMP-9/TNF-α/Gr-1/MCP-1/DUSP-1/SIRT5/VDAC1 and to assess the severity of inflammatory response and the expression of the key target proteins, SIRT5/DUSP1/VDAC, in each group. (Q) Heatmap of gene expression in different cell clusters (R–S) GO/GSEA enrichment analysis of hallmark gene function in each cell cluster to validate the mechanism of action of Ginsenoside Rb1. ∗ represents P < 0.05 and n = 6.

Article Snippet: SIRT5 TG /SIRT5 CKO and NDUFS4 TG /NDUFS4 CKO genetically altered mouse models were bred by Cyagen Biosciences [ ].

Techniques: Sequencing, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rb1 attenuates coronary microvascular inflammatory injury via NDUFS4-SIRT5-DUSP1-mediated mitochondrial quality control in a murine ischemia-reperfusion model

doi: 10.1016/j.jgr.2025.04.006

Figure Lengend Snippet: Ginsenoside Rb1 synergistically regulates mitochondrial biosynthesis via NDUFS-DUSP1. (A.B) Cellular descending clustering graph (C.E) Differential expression of DUSP1 gene in each cell cluster (D.F) Differential expression of NDUFS4 gene in each cell cluster (G) TUNEL for apoptosis rate (H) RT-PCR for SIRT5 transcription level (I) CCK8 for cell activity (J-L.N) WB for PGC1-α/Nrf-2/Nrf-1 expression level (M) RT-PCR for PGC1-α transcription level (O.Q) Immunofluorescence for Nrf-2 content (P) RT-PCR for Nrf-1 transcription level (R)Mito-tracker detection of mitochondrial morphology; LC3 immunofluorescence assay (S) Fluorescence for TOM20 content (T–V) Complex-1/III/V activity assay. ∗ represents P < 0.05 and n = 6.

Article Snippet: SIRT5 TG /SIRT5 CKO and NDUFS4 TG /NDUFS4 CKO genetically altered mouse models were bred by Cyagen Biosciences [ ].

Techniques: Quantitative Proteomics, TUNEL Assay, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Expressing, Immunofluorescence, Fluorescence

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rb1 attenuates coronary microvascular inflammatory injury via NDUFS4-SIRT5-DUSP1-mediated mitochondrial quality control in a murine ischemia-reperfusion model

doi: 10.1016/j.jgr.2025.04.006

Figure Lengend Snippet: Ginsenoside Rb1 upregulates SIRT5 to inhibit I/R injury-mediated mtUPR and inflammatory changes. (A–D) LDH/Troponin T/BNP/CK-MB levels were measured to assess the degree of inflammation in myocardial tissue in each group (E.G-J) RT-PCR was performed to detect the levels of IL-10/IL-17/IL-1β/MMP-9/TNF-α and to assess the transcriptional levels of inflammatory factors in each group (F) Electron fluoroscopy was performed to assess the endothelial structure of coronary artery microvessels in each group (K-M.O-P) RT-PCT to detect the transcription level of mitochondrial unfolded proteins such as Atf5/Lonp1/HSP10/HSP60/CHOP in each group (N) HE staining to detect the pathological damage of myocardium in each group and immunofluorescence to quantitatively detect the expression of apoptotic factor Caspase-9. (Q) Detection of immunofluorescence expression of inflammatory factor Gr-1 in each group (R–S) RT-PCR detection of MCP-1/mtDNA/ClpP/DUSP-1/SIRT5/VDAC-1 transcript levels in each group to assess the transcript levels of mitochondrial unfolded protein response-related regulators, inflammatory factors, and key target proteins. (T) RT-PCR was performed to detect the transcription level of the gene after SIRT5/DUSP1/NDUFS4 knockdown; Western blot was performed to detect the protein expression level after SIRT5/DUSP1/NDUFS4 knockdown. ∗ represents P < 0.05 and n = 6.

Article Snippet: SIRT5 TG /SIRT5 CKO and NDUFS4 TG /NDUFS4 CKO genetically altered mouse models were bred by Cyagen Biosciences [ ].

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

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rb1 attenuates coronary microvascular inflammatory injury via NDUFS4-SIRT5-DUSP1-mediated mitochondrial quality control in a murine ischemia-reperfusion model

doi: 10.1016/j.jgr.2025.04.006

Figure Lengend Snippet: Ginsenoside Rb1 up-regulates SIRT5 to maintain ‘mitochondrial dynamics-autophagy’ dynamic balance. (A) PINK/Parkin/ATG-5 transcript levels (B) Drp-1/Mfn1/OPA-1 transcript levels (C) TUNEL assay for apoptosis (D) CCK-8 assay for cellular activity (E) LC-3 fluorescence detection; Mito-tracker mitochondrial red fluorescence labelling (F) HSP10/HSP60/Lonp-1/CHOP/mtDNA/ClpP transcript levels (G) Mito-tracker mitochondrial green fluorescent labelling (H) Atf5 transcript levels (I.J) Nrf-2 immunofluorescence expression (K) Schematic diagram of mitochondrial dynamics-autophagy (L) PGC1-α/Nrf-2/Tfam transcript levels. ∗ represents P < 0.05 and n = 6.

Article Snippet: SIRT5 TG /SIRT5 CKO and NDUFS4 TG /NDUFS4 CKO genetically altered mouse models were bred by Cyagen Biosciences [ ].

Techniques: TUNEL Assay, CCK-8 Assay, Activity Assay, Fluorescence, Immunofluorescence, Expressing

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rb1 attenuates coronary microvascular inflammatory injury via NDUFS4-SIRT5-DUSP1-mediated mitochondrial quality control in a murine ischemia-reperfusion model

doi: 10.1016/j.jgr.2025.04.006

Figure Lengend Snippet: Ginsenoside Rb1 regulates mitochondrial homeostasis-cellular inflammatory response through SIRT5-DUSP1. (A) Molecular docking of molecular targets of Ginsenoside Rb1 (B) CCK8 assay for cellular activity (C) RT-PCR for apoptotic and inflammatory factors Caspase-1/Caspase-4/IL-1β transcript levels (D) Inflammatory vesicle NLRP3 transcript level assay (E.F) TUNEL assay for apoptosis rate (G) Detection of Mitochondrial functional protein Complex-1/III/V/PGC1-α/Nrf-2/Tfam activity (H) Immunofluorescence assay for Nrf-2 content (I) RT-PCR assay for VDAC-1 transcription level (J) mPTP opening rate assay (K–M) JC-1 mitochondrial membrane potential fluorescence assay. ∗ represents P < 0.05 and n = 6.

Article Snippet: SIRT5 TG /SIRT5 CKO and NDUFS4 TG /NDUFS4 CKO genetically altered mouse models were bred by Cyagen Biosciences [ ].

Techniques: CCK-8 Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, TUNEL Assay, Functional Assay, Immunofluorescence, Membrane, Fluorescence

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rb1 attenuates coronary microvascular inflammatory injury via NDUFS4-SIRT5-DUSP1-mediated mitochondrial quality control in a murine ischemia-reperfusion model

doi: 10.1016/j.jgr.2025.04.006

Figure Lengend Snippet: Ginsenoside Rb1 synergistically regulates the mitochondrial quality control network via SIRT5-NDUFS. (A) CCK8 assay for cellular activity (B) Drp-1/Fis-1/Opa-1/Mfn-1/Mfn-2 transcript levels (C) JC-1 mitochondrial membrane potential assay (D) LC3 immunofluorescence assay, Mito-tracker mitochondrial fluorescence assay (E) RT-PCR assay for the mitochondrial unfolded protein factor HSP10/HSP60/Lonp-1/mtDNAj/CHOP/VDAC-1 (F) Western blot for pan-apoptotic factor AIM2/Pynin/ZBP-1/Cas-1 (G) Western blot for pan-apoptotic factor Cas-8/NLRP3/MLKL/RIPK3/GSDMD/Bax protein expression. ∗ represents P < 0.05 and n = 6.

Article Snippet: SIRT5 TG /SIRT5 CKO and NDUFS4 TG /NDUFS4 CKO genetically altered mouse models were bred by Cyagen Biosciences [ ].

Techniques: Control, CCK-8 Assay, Activity Assay, Membrane, Immunofluorescence, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing