Review





Similar Products

92
ATCC paper emdb emd 25488 sars2 57 vh sequence
Paper Emdb Emd 25488 Sars2 57 Vh Sequence, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/paper emdb emd 25488 sars2 57 vh sequence/product/ATCC
Average 92 stars, based on 1 article reviews
paper emdb emd 25488 sars2 57 vh sequence - by Bioz Stars, 2026-02
92/100 stars
  Buy from Supplier

92
ATCC atcc 25488 t
Atcc 25488 T, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/atcc 25488 t/product/ATCC
Average 92 stars, based on 1 article reviews
atcc 25488 t - by Bioz Stars, 2026-02
92/100 stars
  Buy from Supplier

92
Addgene inc sirt5 locus
A. Gain of extra <t>SIRT5</t> copies in melanoma. BRAF , NRAS , PTEN , MITF , NF1 and other sirtuins are shown for comparison (n=287; data from TCGA, Provisional, analyzed on cBioPortal). ND, not determined. Percentage of samples with any genomic alteration (Any) or amplification or gain (Amp/Gain) is indicated. Graphed are any alterations queried for the indicated gene. Copy number gain indicates a low-level gain of a single additional copy, and amplification refers to high-level amplification (multiple extra copies). Results from the query ( GENE : MUT AMP HOMDEL GAIN HETLOSS) in cBioPortal were analyzed and plotted. B. Kaplan–Meier analysis of overall survival in melanoma patients with or without copy number gain or amplification of SIRT5 . Overall survival was analyzed using the query: “ SIRT5 : AMP GAIN.” C . SIRT5 (6p23) and centromere 6p (Cen6p) amplification (amp) or co-amplification (Co-amp) in melanoma, as assayed by FISH staining (n=32). D. Sirtuin gene copy number (CN) in human melanoma samples, as assayed by high density SNP array (n=139). E. SIRT5 mRNA expression levels in melanoma correlate with Clark’s level (p=0.0044, linear regression; p=0.037, ANOVA). F . SIRT5 protein levels are increased in melanoma relative to benign melanocytic lesions (p=0.0333, Chi-squared; n=14 nevi, n=87 melanoma). See also Figure S1 and Table S1.
Sirt5 Locus, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sirt5 locus/product/Addgene inc
Average 92 stars, based on 1 article reviews
sirt5 locus - by Bioz Stars, 2026-02
92/100 stars
  Buy from Supplier

92
ATCC thermofisher cat
A. Gain of extra <t>SIRT5</t> copies in melanoma. BRAF , NRAS , PTEN , MITF , NF1 and other sirtuins are shown for comparison (n=287; data from TCGA, Provisional, analyzed on cBioPortal). ND, not determined. Percentage of samples with any genomic alteration (Any) or amplification or gain (Amp/Gain) is indicated. Graphed are any alterations queried for the indicated gene. Copy number gain indicates a low-level gain of a single additional copy, and amplification refers to high-level amplification (multiple extra copies). Results from the query ( GENE : MUT AMP HOMDEL GAIN HETLOSS) in cBioPortal were analyzed and plotted. B. Kaplan–Meier analysis of overall survival in melanoma patients with or without copy number gain or amplification of SIRT5 . Overall survival was analyzed using the query: “ SIRT5 : AMP GAIN.” C . SIRT5 (6p23) and centromere 6p (Cen6p) amplification (amp) or co-amplification (Co-amp) in melanoma, as assayed by FISH staining (n=32). D. Sirtuin gene copy number (CN) in human melanoma samples, as assayed by high density SNP array (n=139). E. SIRT5 mRNA expression levels in melanoma correlate with Clark’s level (p=0.0044, linear regression; p=0.037, ANOVA). F . SIRT5 protein levels are increased in melanoma relative to benign melanocytic lesions (p=0.0333, Chi-squared; n=14 nevi, n=87 melanoma). See also Figure S1 and Table S1.
Thermofisher Cat, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/thermofisher cat/product/ATCC
Average 92 stars, based on 1 article reviews
thermofisher cat - by Bioz Stars, 2026-02
92/100 stars
  Buy from Supplier

92
Addgene inc murine sirt5 promoter
A. Gain of extra <t>SIRT5</t> copies in melanoma. BRAF , NRAS , PTEN , MITF , NF1 and other sirtuins are shown for comparison (n=287; data from TCGA, Provisional, analyzed on cBioPortal). ND, not determined. Percentage of samples with any genomic alteration (Any) or amplification or gain (Amp/Gain) is indicated. Graphed are any alterations queried for the indicated gene. Copy number gain indicates a low-level gain of a single additional copy, and amplification refers to high-level amplification (multiple extra copies). Results from the query ( GENE : MUT AMP HOMDEL GAIN HETLOSS) in cBioPortal were analyzed and plotted. B. Kaplan–Meier analysis of overall survival in melanoma patients with or without copy number gain or amplification of SIRT5 . Overall survival was analyzed using the query: “ SIRT5 : AMP GAIN.” C . SIRT5 (6p23) and centromere 6p (Cen6p) amplification (amp) or co-amplification (Co-amp) in melanoma, as assayed by FISH staining (n=32). D. Sirtuin gene copy number (CN) in human melanoma samples, as assayed by high density SNP array (n=139). E. SIRT5 mRNA expression levels in melanoma correlate with Clark’s level (p=0.0044, linear regression; p=0.037, ANOVA). F . SIRT5 protein levels are increased in melanoma relative to benign melanocytic lesions (p=0.0333, Chi-squared; n=14 nevi, n=87 melanoma). See also Figure S1 and Table S1.
Murine Sirt5 Promoter, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/murine sirt5 promoter/product/Addgene inc
Average 92 stars, based on 1 article reviews
murine sirt5 promoter - by Bioz Stars, 2026-02
92/100 stars
  Buy from Supplier

92
ATCC atcc 25488
A. Gain of extra <t>SIRT5</t> copies in melanoma. BRAF , NRAS , PTEN , MITF , NF1 and other sirtuins are shown for comparison (n=287; data from TCGA, Provisional, analyzed on cBioPortal). ND, not determined. Percentage of samples with any genomic alteration (Any) or amplification or gain (Amp/Gain) is indicated. Graphed are any alterations queried for the indicated gene. Copy number gain indicates a low-level gain of a single additional copy, and amplification refers to high-level amplification (multiple extra copies). Results from the query ( GENE : MUT AMP HOMDEL GAIN HETLOSS) in cBioPortal were analyzed and plotted. B. Kaplan–Meier analysis of overall survival in melanoma patients with or without copy number gain or amplification of SIRT5 . Overall survival was analyzed using the query: “ SIRT5 : AMP GAIN.” C . SIRT5 (6p23) and centromere 6p (Cen6p) amplification (amp) or co-amplification (Co-amp) in melanoma, as assayed by FISH staining (n=32). D. Sirtuin gene copy number (CN) in human melanoma samples, as assayed by high density SNP array (n=139). E. SIRT5 mRNA expression levels in melanoma correlate with Clark’s level (p=0.0044, linear regression; p=0.037, ANOVA). F . SIRT5 protein levels are increased in melanoma relative to benign melanocytic lesions (p=0.0333, Chi-squared; n=14 nevi, n=87 melanoma). See also Figure S1 and Table S1.
Atcc 25488, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/atcc 25488/product/ATCC
Average 92 stars, based on 1 article reviews
atcc 25488 - by Bioz Stars, 2026-02
92/100 stars
  Buy from Supplier

Image Search Results


A. Gain of extra SIRT5 copies in melanoma. BRAF , NRAS , PTEN , MITF , NF1 and other sirtuins are shown for comparison (n=287; data from TCGA, Provisional, analyzed on cBioPortal). ND, not determined. Percentage of samples with any genomic alteration (Any) or amplification or gain (Amp/Gain) is indicated. Graphed are any alterations queried for the indicated gene. Copy number gain indicates a low-level gain of a single additional copy, and amplification refers to high-level amplification (multiple extra copies). Results from the query ( GENE : MUT AMP HOMDEL GAIN HETLOSS) in cBioPortal were analyzed and plotted. B. Kaplan–Meier analysis of overall survival in melanoma patients with or without copy number gain or amplification of SIRT5 . Overall survival was analyzed using the query: “ SIRT5 : AMP GAIN.” C . SIRT5 (6p23) and centromere 6p (Cen6p) amplification (amp) or co-amplification (Co-amp) in melanoma, as assayed by FISH staining (n=32). D. Sirtuin gene copy number (CN) in human melanoma samples, as assayed by high density SNP array (n=139). E. SIRT5 mRNA expression levels in melanoma correlate with Clark’s level (p=0.0044, linear regression; p=0.037, ANOVA). F . SIRT5 protein levels are increased in melanoma relative to benign melanocytic lesions (p=0.0333, Chi-squared; n=14 nevi, n=87 melanoma). See also Figure S1 and Table S1.

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: A. Gain of extra SIRT5 copies in melanoma. BRAF , NRAS , PTEN , MITF , NF1 and other sirtuins are shown for comparison (n=287; data from TCGA, Provisional, analyzed on cBioPortal). ND, not determined. Percentage of samples with any genomic alteration (Any) or amplification or gain (Amp/Gain) is indicated. Graphed are any alterations queried for the indicated gene. Copy number gain indicates a low-level gain of a single additional copy, and amplification refers to high-level amplification (multiple extra copies). Results from the query ( GENE : MUT AMP HOMDEL GAIN HETLOSS) in cBioPortal were analyzed and plotted. B. Kaplan–Meier analysis of overall survival in melanoma patients with or without copy number gain or amplification of SIRT5 . Overall survival was analyzed using the query: “ SIRT5 : AMP GAIN.” C . SIRT5 (6p23) and centromere 6p (Cen6p) amplification (amp) or co-amplification (Co-amp) in melanoma, as assayed by FISH staining (n=32). D. Sirtuin gene copy number (CN) in human melanoma samples, as assayed by high density SNP array (n=139). E. SIRT5 mRNA expression levels in melanoma correlate with Clark’s level (p=0.0044, linear regression; p=0.037, ANOVA). F . SIRT5 protein levels are increased in melanoma relative to benign melanocytic lesions (p=0.0333, Chi-squared; n=14 nevi, n=87 melanoma). See also Figure S1 and Table S1.

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Comparison, Amplification, Staining, Expressing

A. BRAF or NRAS mutant melanoma cell lines indicated were infected with a non-targeting shRNA (control) or one of two SIRT5 shRNAs (KD1 or KD2). Equivalent cell numbers were then plated 48 hrs. post-transduction into 96-well plates in the presence of puromycin. Cell mass was determined at the indicated timepoints via WST-1 assay, with absorbance measured at 450nm. Average results (n=6/timepoint) are graphed. Error bars represent standard deviation. Representative of 5/5 SIRT5 shRNAs tested (see also ). B. SIRT5 KD results in significantly (p<0.0001, unpaired Student’s t-test) impaired colony formation by A2058 and SK-MEL-2 cells 12 days post-transduction. Cell mass was assayed using crystal violet staining, with absorbance measured at 590nm. Average of n=12 technical replicates results are plotted. Error bars represent SD. Representative crystal violet-stained wells are shown. Lower panel, representative immunoblot analysis demonstrating SIRT5 KD. C. Top panel, viability of A2058 cells transfected with the indicated CRISPR guide RNA (Control or G1-G4). Cell mass was assayed using crystal violet staining, with absorbance measured at 590nm. Average of n=9 technical replicates results are plotted. Error bars represent standard deviation. Significance calculated using unpaired Student’s t-test. Bottom panel, representative immunoblot analysis confirming CRISPR-mediated SIRT5 loss (Control: empty vector).

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: A. BRAF or NRAS mutant melanoma cell lines indicated were infected with a non-targeting shRNA (control) or one of two SIRT5 shRNAs (KD1 or KD2). Equivalent cell numbers were then plated 48 hrs. post-transduction into 96-well plates in the presence of puromycin. Cell mass was determined at the indicated timepoints via WST-1 assay, with absorbance measured at 450nm. Average results (n=6/timepoint) are graphed. Error bars represent standard deviation. Representative of 5/5 SIRT5 shRNAs tested (see also ). B. SIRT5 KD results in significantly (p<0.0001, unpaired Student’s t-test) impaired colony formation by A2058 and SK-MEL-2 cells 12 days post-transduction. Cell mass was assayed using crystal violet staining, with absorbance measured at 590nm. Average of n=12 technical replicates results are plotted. Error bars represent SD. Representative crystal violet-stained wells are shown. Lower panel, representative immunoblot analysis demonstrating SIRT5 KD. C. Top panel, viability of A2058 cells transfected with the indicated CRISPR guide RNA (Control or G1-G4). Cell mass was assayed using crystal violet staining, with absorbance measured at 590nm. Average of n=9 technical replicates results are plotted. Error bars represent standard deviation. Significance calculated using unpaired Student’s t-test. Bottom panel, representative immunoblot analysis confirming CRISPR-mediated SIRT5 loss (Control: empty vector).

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Mutagenesis, Infection, shRNA, Control, Transduction, WST-1 Assay, Standard Deviation, Staining, Western Blot, Transfection, CRISPR, Plasmid Preparation

A. Immunoblot analysis demonstrating induction of caspase 3 cleavage 72 and 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) in A2058 and SK-MEL-2 cell lines. B . Viability of MP-41, A2058 or YUMM5.2 cells infected with control (C) or one of five SIRT5 shRNAs (KD1-KD5) against human SIRT5 (top and middle panels) or mouse Sirt5 (bottom panel). Average results (n=6/timepoint) are graphed. Error bars represent standard deviation. Right panels: immunoblot analysis demonstrating loss of SIRT5 and induction of caspase 3 cleavage following SIRT5 KD. C. Flow cytometric analysis of A2058 cells stained with Annexin V and propidium iodide (PI), as indicated, showing an increased fraction of Annexin V-positive cells 96 hrs. after SIRT5 KD. D. Average of n=3 technical replicates is plotted. Error bars represent (SD). Significance calculated using unpaired Student’s t-test. Increased Annexin V + staining is observed in both the PI-positive and PI-negative populations.

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: A. Immunoblot analysis demonstrating induction of caspase 3 cleavage 72 and 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) in A2058 and SK-MEL-2 cell lines. B . Viability of MP-41, A2058 or YUMM5.2 cells infected with control (C) or one of five SIRT5 shRNAs (KD1-KD5) against human SIRT5 (top and middle panels) or mouse Sirt5 (bottom panel). Average results (n=6/timepoint) are graphed. Error bars represent standard deviation. Right panels: immunoblot analysis demonstrating loss of SIRT5 and induction of caspase 3 cleavage following SIRT5 KD. C. Flow cytometric analysis of A2058 cells stained with Annexin V and propidium iodide (PI), as indicated, showing an increased fraction of Annexin V-positive cells 96 hrs. after SIRT5 KD. D. Average of n=3 technical replicates is plotted. Error bars represent (SD). Significance calculated using unpaired Student’s t-test. Increased Annexin V + staining is observed in both the PI-positive and PI-negative populations.

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Western Blot, Transduction, Infection, Control, Standard Deviation, Staining

A. SIRT5 depletion in A2058 cells results in attenuated xenograft tumor growth. Quantification of tumor size was initiated on day 13 after initial injection of cells (left panel). Tumor size was recorded with Vernier calipers on the days indicated. Each point represents the measurements on n=5 mice for each condition (C, KD1, or KD2). Pairwise representation of endpoint tumor size in each mouse within each group is plotted (right panel). Average tumor mass measurements at day 28 are plotted (p<0.05, paired two-tailed t-test for each group). Error bars represent SD. B. Mice were sacrificed, and tumors were dissected at 28 days after initial injection. Scale bar below tumors=2cm. C . SIRT5 deficiency attenuates tumor formation in an autochthonous melanoma model. Sirt5 deficient-mice were bred into the Braf CA ;Pten fl/fl ;Tyr::CreER background . Melanomas were induced in littermate male Sirt5 WT or Sirt5 KO mice as shown by topical application of 4HT at ages 4-9 weeks; tumors were weighed following euthanasia. Averages of 5 sets of male mice are plotted (p<0.05, paired two-tailed t-test). Means ± SD are shown. D. SIRT5 immunoblot of a representative tumor from a Sirt5 WT or KO male or female mouse (left panel). Representative tumor from a Sirt5 WT or KO male mouse, as indicated, after 4HT induction (right panel). Scale bar=1cm.

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: A. SIRT5 depletion in A2058 cells results in attenuated xenograft tumor growth. Quantification of tumor size was initiated on day 13 after initial injection of cells (left panel). Tumor size was recorded with Vernier calipers on the days indicated. Each point represents the measurements on n=5 mice for each condition (C, KD1, or KD2). Pairwise representation of endpoint tumor size in each mouse within each group is plotted (right panel). Average tumor mass measurements at day 28 are plotted (p<0.05, paired two-tailed t-test for each group). Error bars represent SD. B. Mice were sacrificed, and tumors were dissected at 28 days after initial injection. Scale bar below tumors=2cm. C . SIRT5 deficiency attenuates tumor formation in an autochthonous melanoma model. Sirt5 deficient-mice were bred into the Braf CA ;Pten fl/fl ;Tyr::CreER background . Melanomas were induced in littermate male Sirt5 WT or Sirt5 KO mice as shown by topical application of 4HT at ages 4-9 weeks; tumors were weighed following euthanasia. Averages of 5 sets of male mice are plotted (p<0.05, paired two-tailed t-test). Means ± SD are shown. D. SIRT5 immunoblot of a representative tumor from a Sirt5 WT or KO male or female mouse (left panel). Representative tumor from a Sirt5 WT or KO male mouse, as indicated, after 4HT induction (right panel). Scale bar=1cm.

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Injection, Two Tailed Test, Western Blot

A2058 and A375 cells maintain glycolytic function ( A. ), glucose-dependent mitochondrial respiration ( B .), and ATP production ( C. ) upon SIRT5 depletion compared to control cells. Mitochondrial respiration, glycolytic stress tests, and ATP production rates were measured at 72 hrs. post-transduction with shRNAs against SIRT5 using a Seahorse XFe96 Analyzer. All rates are normalized to total protein content per sample (n=6 for A. and C. , n=5 for B. ). OCR, oxygen consumption rate; ECAR, extracellular acidification rate. Error bars represent standard deviation. D. Mitochondrial membrane potential is stable in A2058 cells after SIRT5 loss (C, control cells, n=6). Cells were incubated with JC-1, a dye which exhibits membrane potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green to red. Mitochondrial depolarization is indicated by a decrease in the red:green (aggregate:monomer) fluorescence intensity ratio. FCCP, a mitochondrial uncoupler, depolarizes mitochondrial membrane potential and is used as a positive control. Significance calculated using unpaired Student’s t-test.

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: A2058 and A375 cells maintain glycolytic function ( A. ), glucose-dependent mitochondrial respiration ( B .), and ATP production ( C. ) upon SIRT5 depletion compared to control cells. Mitochondrial respiration, glycolytic stress tests, and ATP production rates were measured at 72 hrs. post-transduction with shRNAs against SIRT5 using a Seahorse XFe96 Analyzer. All rates are normalized to total protein content per sample (n=6 for A. and C. , n=5 for B. ). OCR, oxygen consumption rate; ECAR, extracellular acidification rate. Error bars represent standard deviation. D. Mitochondrial membrane potential is stable in A2058 cells after SIRT5 loss (C, control cells, n=6). Cells were incubated with JC-1, a dye which exhibits membrane potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green to red. Mitochondrial depolarization is indicated by a decrease in the red:green (aggregate:monomer) fluorescence intensity ratio. FCCP, a mitochondrial uncoupler, depolarizes mitochondrial membrane potential and is used as a positive control. Significance calculated using unpaired Student’s t-test.

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Control, Transduction, Standard Deviation, Membrane, Incubation, Fluorescence, Positive Control

Genes ( A. ) upregulated or ( B. ) downregulated upon SIRT5 KD. Only genes significantly (p<0.05) altered in both KDs in each cell line, as indicated, were scored. C. Expression levels of differentially expressed genes (DEGs; qadj<0.05) in response to SIRT5 KD were correlated with SIRT5 gene expression using Spearman’s rank correlation coefficient in 443 sequenced human skin cutaneous melanoma (SKCM) samples, identifying DEGs with significant clinical correlation with SIRT5 expression (q<0.01). Labeled genes represent oncogenes or extremely correlated genes most significantly altered by SIRT5 knockdown (q<0.0001, log2FoldChange>2). D. Immunoblot demonstrating loss of MITF expression 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C) in 5 cutaneous and one uveal melanoma cell lines, as indicated. E. Relative FPKMs in A2058 and SK-MEL-2 cells demonstrate a loss of MITF (bar graphs, upper panels) and several MITF target gene transcripts upon SIRT5 KD (heatmaps, lower panels). Scale bars adjacent to heat maps indicate linear fold change (control (C) set to 1). Significance calculated using unpaired Student’s t-test. F . Expression of SIRT5 , MITF and the MITF target, PPARGC1A are positively correlated in melanoma clinical samples (p<0.0001, data from TCGA, analyzed on cBioPortal; see ).

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: Genes ( A. ) upregulated or ( B. ) downregulated upon SIRT5 KD. Only genes significantly (p<0.05) altered in both KDs in each cell line, as indicated, were scored. C. Expression levels of differentially expressed genes (DEGs; qadj<0.05) in response to SIRT5 KD were correlated with SIRT5 gene expression using Spearman’s rank correlation coefficient in 443 sequenced human skin cutaneous melanoma (SKCM) samples, identifying DEGs with significant clinical correlation with SIRT5 expression (q<0.01). Labeled genes represent oncogenes or extremely correlated genes most significantly altered by SIRT5 knockdown (q<0.0001, log2FoldChange>2). D. Immunoblot demonstrating loss of MITF expression 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C) in 5 cutaneous and one uveal melanoma cell lines, as indicated. E. Relative FPKMs in A2058 and SK-MEL-2 cells demonstrate a loss of MITF (bar graphs, upper panels) and several MITF target gene transcripts upon SIRT5 KD (heatmaps, lower panels). Scale bars adjacent to heat maps indicate linear fold change (control (C) set to 1). Significance calculated using unpaired Student’s t-test. F . Expression of SIRT5 , MITF and the MITF target, PPARGC1A are positively correlated in melanoma clinical samples (p<0.0001, data from TCGA, analyzed on cBioPortal; see ).

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Expressing, Gene Expression, Labeling, Knockdown, Western Blot, Transduction, Control

A. Heatmap of z-scores calculated from metabolic reaction fluxes predicted by genome-scale modeling to be differentially active (p<0.01) after SIRT5 KD. B. Total histone acetylation is reduced 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C) in melanoma cell lines. Lanes were run on the same gel but are noncontiguous. C. Immunoblot demonstrating loss of H3K9ac and H4K16ac 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C) in A2058 cells. D. H3K9ac is reduced within the promoter regions of MITF and c-Myc in SIRT5-depleted A2058 cells via CUT&RUN followed by qRT-PCR. Signal (Ct values) relative to input DNA were normalized to control (C) samples for each primer set. Graphed are averages of n=9 replicates. Error bars represent SD. Significance calculated using unpaired Student’s t-test. Acetylation ( E. ) and MITF expression ( F. ) are restored in A2058 cells lacking SIRT5 after 4 weeks of continual culture in puromycin. G. Total cellular acetyl-CoA levels are increased in A2058, A375 and SK-MEL-2 cells 96 hrs. after SIRT5 depletion. Acetyl-CoA abundance was quantified by liquid chromatography-high resolution mass spectrometry and normalized to cell number. Plotted are average (n=5) acetyl-CoA levels as pmol acetyl-CoA/10 5 cells. Error bars represent SD. Significance calculated using unpaired Student’s t-test.

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: A. Heatmap of z-scores calculated from metabolic reaction fluxes predicted by genome-scale modeling to be differentially active (p<0.01) after SIRT5 KD. B. Total histone acetylation is reduced 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C) in melanoma cell lines. Lanes were run on the same gel but are noncontiguous. C. Immunoblot demonstrating loss of H3K9ac and H4K16ac 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C) in A2058 cells. D. H3K9ac is reduced within the promoter regions of MITF and c-Myc in SIRT5-depleted A2058 cells via CUT&RUN followed by qRT-PCR. Signal (Ct values) relative to input DNA were normalized to control (C) samples for each primer set. Graphed are averages of n=9 replicates. Error bars represent SD. Significance calculated using unpaired Student’s t-test. Acetylation ( E. ) and MITF expression ( F. ) are restored in A2058 cells lacking SIRT5 after 4 weeks of continual culture in puromycin. G. Total cellular acetyl-CoA levels are increased in A2058, A375 and SK-MEL-2 cells 96 hrs. after SIRT5 depletion. Acetyl-CoA abundance was quantified by liquid chromatography-high resolution mass spectrometry and normalized to cell number. Plotted are average (n=5) acetyl-CoA levels as pmol acetyl-CoA/10 5 cells. Error bars represent SD. Significance calculated using unpaired Student’s t-test.

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Transduction, Control, Western Blot, Quantitative RT-PCR, Expressing, Liquid Chromatography, Mass Spectrometry

A. LC-MS/MS-based metabolite profiling followed by MetaboAnalyst pathway analysis demonstrate alterations in glycine and serine and methionine biosynthesis pathways in melanoma cells upon SIRT5 depletion. B. Perturbations in 1C metabolite levels in response to SIRT5 loss in the cell lines shown. Each column represents the mean of 3 independently prepared biological replicates. Metabolite levels in SIRT5 depleted (KD1 and KD2, as indicated) samples are normalized to control. SAM, S-adenosyl-methionine; SAH, S-adenosylhomocysteine; GSH, reduced glutathione; GSSG, glutathione disulfide. C. H3K4me3 and H3K9me3 immunoblot in melanoma cells 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C). D. H3K4me3 and H3K9me3 levels are restored in A2058 cells lacking SIRT5 after 4 weeks of continual culture in puromycin. E. Flow cytometric analysis of DCFDA-stained A2058 cells 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) reveals increased ROS compared to a non-targeting control (C), p<0.005. Left panel, average mean fluorescence intensity of DCFDA positive populations in n=3 samples. Error bars represent SD. Significance calculated using unpaired Student’s t-test. Right panel, representative flow cytometric of A2058 cells stained with DCFDA. F. SIRT5 interacts with MTHFD1L in A2058 cells. Increasing amounts of anti-SIRT5 antibody increases SIRT5-MTHFD1L coprecipitation compared to normal rabbit IgG control. Basal expression of SIRT5 and MTHFD1L in whole-cell extract (1% of initial amount used for immunoprecipitation) is shown for comparison. G. Proposed model of promotion of MITF and c-MYC expression via SIRT5-dependent chromatin modifications in human melanoma. Me, methylation; Ac, acetylation.

Journal: bioRxiv

Article Title: The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

doi: 10.1101/2020.09.07.286526

Figure Lengend Snippet: A. LC-MS/MS-based metabolite profiling followed by MetaboAnalyst pathway analysis demonstrate alterations in glycine and serine and methionine biosynthesis pathways in melanoma cells upon SIRT5 depletion. B. Perturbations in 1C metabolite levels in response to SIRT5 loss in the cell lines shown. Each column represents the mean of 3 independently prepared biological replicates. Metabolite levels in SIRT5 depleted (KD1 and KD2, as indicated) samples are normalized to control. SAM, S-adenosyl-methionine; SAH, S-adenosylhomocysteine; GSH, reduced glutathione; GSSG, glutathione disulfide. C. H3K4me3 and H3K9me3 immunoblot in melanoma cells 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) compared to a non-targeting control (C). D. H3K4me3 and H3K9me3 levels are restored in A2058 cells lacking SIRT5 after 4 weeks of continual culture in puromycin. E. Flow cytometric analysis of DCFDA-stained A2058 cells 96 hrs. post-transduction with shRNAs targeting SIRT5 (KD1 or KD2) reveals increased ROS compared to a non-targeting control (C), p<0.005. Left panel, average mean fluorescence intensity of DCFDA positive populations in n=3 samples. Error bars represent SD. Significance calculated using unpaired Student’s t-test. Right panel, representative flow cytometric of A2058 cells stained with DCFDA. F. SIRT5 interacts with MTHFD1L in A2058 cells. Increasing amounts of anti-SIRT5 antibody increases SIRT5-MTHFD1L coprecipitation compared to normal rabbit IgG control. Basal expression of SIRT5 and MTHFD1L in whole-cell extract (1% of initial amount used for immunoprecipitation) is shown for comparison. G. Proposed model of promotion of MITF and c-MYC expression via SIRT5-dependent chromatin modifications in human melanoma. Me, methylation; Ac, acetylation.

Article Snippet: Briefly, guide sequences (Table S6) targeting the SIRT5 locus were inserted into pSpCas9(BB)-2A-Puro (PX459) backbone (Addgene catalog #62988).

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Western Blot, Transduction, Staining, Fluorescence, Expressing, Immunoprecipitation, Comparison, Methylation