Journal: Scientific Reports

Article Title: Type 2 Innate Lymphocytes Actuate Immunity Against Tumours and Limit Cancer Metastasis

doi: 10.1038/s41598-018-20608-6

Figure Lengend Snippet: Genetic complementation of immune evasive tumours shows a clear phenotypic shift towards immune recognition. Immunohistochemical staining was used to visualise tumour infiltrating immune cells. CD4 is a glycoprotein found on the surface of T helper cells, macrophages, dendritic cells. CD8 is primarily a marker for cytotoxic T cells, but also found on natural killer cells and DCs; CD68 is a marker of monocytes and macrophages; Ly6G is a marker for neutrophils; FoxP3 is a marker of regulatory T cells. Greater numbers of CD4 + and CD8 + cells can be seen within ( c , g ) the genetically modified (metastatic A9+IL-33) tumours versus unmodified metastatic (A9) tumours ( a , e ). Fewer regulatory T cells are present in IL-33 expressing tumours, as indicated by lower FoxP3 staining; metastatic A9 ( q ) versus metastatic A9+IL-33 ( s ) or primary TC1 ( r ) tumours. Increased macrophage and neutrophil responses are seen in IL-33 expressing tumours: unmodified metastatic A9 ( i , m ) versus metastatic A9+IL-33 ( k , o ) or primary TC1 ( j , n ) tumours respectively. ( d , h , l , p , t ) Negative controls (rat IgG targeting Keyhole Limpet Hemocyanin (KLH)) were included to show that non-specific staining was minimized. The percentage of positively stained cells was calculated from the total number of cells on each slide. 10 µm thick sections were stained with appropriate antibodies and imaged at 20X magnification ( a – t ); size bar = 100 µm ( a – t ).

Article Snippet: CD8 + dendritic cells (DC) were isolated from C57Bl/6 mouse using the CD8 + DC Isolation kit for mouse (130-091-169, Miltenyi Biotec, Inc.).

Techniques: Immunohistochemical staining, Staining, Marker, Genetically Modified, Expressing

Journal: Scientific Reports

Article Title: Type 2 Innate Lymphocytes Actuate Immunity Against Tumours and Limit Cancer Metastasis

doi: 10.1038/s41598-018-20608-6

Figure Lengend Snippet: The mice lacking ILC2s (RORα −/− ) were less able to limit the growth of tumours. Tumours with and without IL-33-expression were established in the WT and RORα −/− chimeric mice. ( a ) The presence of ILC2s significantly inhibited tumour formation in WT chimeric mice bearing tumours expressing IL-33, when compared to RORα −/− chimeras, which lacked ILC2s. Metastatic A9 tumours demonstrated rapid progression and severe disease symptoms reaching the humane end-point that resulted in early termination of animals from both A9 chimeric groups, indicating that the ILC2s were not significantly able to control the growth of fast growing tumours which lack IL-33 expression. Metastatic A9+IL-33 tumour growth was more aggressive in RORα −/− chimeric mice lacking ILC2s, to the point where they resembled the growth of A9 cells alone. The difference in growth results between metastatic A9+IL-33 tumours grown in WT versus RORα −/− chimeric mice was significant; P < 0.002 (Student t-test). Primary TC1 tumours were able to grow faster in mice lacking ILC2s (RORα −/− chimeras) than in WT chimeras P < 0.05 (Student t-test). ( b ) The numbers of ILC2 cells found in neighbouring lymph nodes were significantly lower in RORα −/− mice compared to WT chimeras bearing IL-33 expressing tumours. This served as a quality control for bone marrow transplantation; *P < 0.05 (Student t-test). ( c ) The numbers of ILC2 cells found in primary TC1 tumours were significantly higher than in metastatic A9 or A9+IL-33 tumours; *P < 0.05 (Student t-test). ( d,e ) RORα deficiency had no effect on the percentage of CD4 + or CD8 + lymphocytes found in either lymph nodes or tumours in response to ( d ) primary TC1 or ( e ) metastatic A9+IL-33 tumours. The percentage of all cells was calculated as a fraction of 2 × 10 5 cellular events used to create a profile for each organ or tissue. The error bars represent standard error of the mean; n = 8 mice per group.

Article Snippet: CD8 + dendritic cells (DC) were isolated from C57Bl/6 mouse using the CD8 + DC Isolation kit for mouse (130-091-169, Miltenyi Biotec, Inc.).

Techniques: Expressing, Control, Transplantation Assay

Journal: Scientific Reports

Article Title: Type 2 Innate Lymphocytes Actuate Immunity Against Tumours and Limit Cancer Metastasis

doi: 10.1038/s41598-018-20608-6

Figure Lengend Snippet: Impact of ILC2s on specific CTL effector mechanisms. Co-culture of metastatic murine prostate TAP-1-low carcinoma cells (LMD) and CD8 T cells with ILC2s (right panel) or without ILC2 cells (left panel): ( a ) TAP-1 expression level in LMD cells after activation with ILC2 cells (right) and without ILC2 cells (left); ( b ) Granzyme b expression by CTL cells in the absence of ILC2s (left) or in the presence of ILC2s (right); ( c ) Light microscope images of cell co-cultures after the incubation with CTLs show the extent of CTL-mediated killing, either in the presence or absence of ILC2s.

Article Snippet: CD8 + dendritic cells (DC) were isolated from C57Bl/6 mouse using the CD8 + DC Isolation kit for mouse (130-091-169, Miltenyi Biotec, Inc.).

Techniques: Co-Culture Assay, Expressing, Activation Assay, Light Microscopy, Incubation

Journal: Scientific Reports

Article Title: Type 2 Innate Lymphocytes Actuate Immunity Against Tumours and Limit Cancer Metastasis

doi: 10.1038/s41598-018-20608-6

Figure Lengend Snippet: Model linking the adaptive and innate immune responses during tumour development via IL-33-ILC2 axis. IL-33-expressing tumour environment stimulates the development of ILC2 cells and functionally activates them through the ST2 receptor pathway. Functionally active ILC2s alter the tumour microenvironment triggering both innate and adaptive immune responses. ILC2s recruit DCs through IL-13 production, stimulate Th2 cells and possibly Th1 cells through direct ILC2 antigen presentation via MHC-II molecules, and indirectly stimulate CTL precursor cells through DC endogenous antigen presentation or cross-presentation via MHC-I molecules. Th1 and Th2 cells may also be activated by DCs through exogenous antigen presentation via MHC-II molecules. Through the release of IL-5 by ILC2s and subsequent recruitment of eosinophils, the chemokine profiles of tumour microenvironment is changed to attract Th1 and CD8+ T cells and to direct the activation of CTL-mediated killing and cancer rejection. This mechanism acts to suppress the frequency of circulating tumours cell and subsequent metastasis. In metastatic tumours with low IL-33 content, the IL-33/ILC2 pathway is not initiated and immune escape is facilitated.

Article Snippet: CD8 + dendritic cells (DC) were isolated from C57Bl/6 mouse using the CD8 + DC Isolation kit for mouse (130-091-169, Miltenyi Biotec, Inc.).

Techniques: Expressing, Immunopeptidomics, Activation Assay

Journal: PLoS ONE

Article Title: CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells

doi: 10.1371/journal.pone.0130519

Figure Lengend Snippet: A. Representative flow cytometry histograms of CaCo-2 cells immunolabeled with anti-AC133 Ab ( left panel ) or anti-CD133CT Ab ( right panel ). B. Quantification of AC133- and CD133CT-positive cells from three independent experiments. Bars represent mean percentage of positive cells ± SEM. C. Immunofluorescence microscopy of CaCo-2 cells co-stained with anti-AC133 Ab (green) and anti-CD133CT Ab (red). Nuclei were counterstained using DAPI (blue). Confocal microscopy images ( inset ) show the subcellular distribution of the staining. Merged image shows overlapping signals generated by anti-AC133 Ab ( green ) and anti-CD133CT Ab ( red ) antibodies. Scale bars correspond to 50 μm (main fields) and 7 μm (insets).

Article Snippet: The human colon carcinoma cell line CaCo-2 [ ] was a kind gift from Dr. Frauke Alvez (University Medical Center, Göttingen) and was obtained from the German collection for microorganims and cell cultures (DSMZ, No. ACC169).

Techniques: Flow Cytometry, Immunolabeling, Immunofluorescence, Microscopy, Staining, Confocal Microscopy, Generated

Journal: PLoS ONE

Article Title: CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells

doi: 10.1371/journal.pone.0130519

Figure Lengend Snippet: A. Western blot analysis of crude lysates (100 μg total protein per lane) from mock-treated CaCo-2 cells (lanes 1) or CaCo-2 cells transfected with the validated anti-CD133 siRNA (LifeTechnologies) (lanes 2–4). After the transfer to the PVDF membrane, Ponceau S staining was done to verify the efficacy of protein transfer ( top panel ) and followed by sequential probing with anti-AC133 Ab ( middle panel ) and anti-CD133CT Ab ( bottom panel ), respectively. B. Comparative assessment of CD133 in unfractionated cell lysates (“input”, 20 μg) and plasma membranes (“PM”, 1 μg) by anti-AC133 or anti-CD133CT Abs.

Article Snippet: The human colon carcinoma cell line CaCo-2 [ ] was a kind gift from Dr. Frauke Alvez (University Medical Center, Göttingen) and was obtained from the German collection for microorganims and cell cultures (DSMZ, No. ACC169).

Techniques: Western Blot, Transfection, Membrane, Staining, Clinical Proteomics

Journal: PLoS ONE

Article Title: CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells

doi: 10.1371/journal.pone.0130519

Figure Lengend Snippet: A. Representative histograms showing CD133 surface expression detected by anti-AC133 Ab ( left panels ) or anti-CD133CT Ab ( right panels ) in primary GSCs cultures and stem-like glioma clone G112SP. B. Mean percentage of cells positively labelled with anti-AC133 Ab ( blue ) or anti-CD133CT Ab ( black) in a panel of GSC lines and CaCo-2 cells used as a positive control. C. Comparative assessment of the total (“input”) and membrane-associated (“PM”) CD133 protein in human GSC lines No. 1063, No. 1080 and No. 1051. 20 μg of proteins were loaded per lane. D. Representative histograms showing surface expression of CD133/2 detected by anti-AC141 Ab in primary cultures of GSCs, stem-like glioma clone G112SP and reference cell line CaCo-2 used as a positive control.

Article Snippet: The human colon carcinoma cell line CaCo-2 [ ] was a kind gift from Dr. Frauke Alvez (University Medical Center, Göttingen) and was obtained from the German collection for microorganims and cell cultures (DSMZ, No. ACC169).

Techniques: Expressing, Positive Control, Membrane

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: (A) Western blot of SDHC protein in SDHC KO and control cells. (B) Transmission electron microscopy analysis of mitochondrial morphology in SDHC KO and control cells. (C) RNA-seq analysis of ADP-ribose-producing biological activities in SDHC KO and control cells. (D) Quantification of NAD + /NADH ratio in SDHC KO and control cells (* denotes p-value < 0.05 by two-sided t-test). (E) SILAC proteomic quantification of LDH and NADH Dehydrogenase complex subunit expression change in SDHC KO cells relative to control. (F) Analysis of polar metabolites involved in glycolysis, fermentation, TCA cycle, aspartate anaplerosis, and glutamine cataplerosis in SDHC KO and control cells. (G) Flow cytometry analysis of sirtuin activity using genetically-encoded reporter. Conditions denoted “+ Plasmids” indicate transfection with pcDNA-mCherry-EGFP (K85TAG) and pCMVAcKRS-tRNAPyl plasmids. “+AcK” denotes the addition of acetyllysine to 5 mM concentration. (H) Western blot analysis of sirtuin protein levels in cytosolic and nuclear subcellular fractions for SDHC KO and control.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Western Blot, Control, Transmission Assay, Electron Microscopy, RNA Sequencing, Multiplex sample analysis, Expressing, Flow Cytometry, Activity Assay, Transfection, Concentration Assay

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: (A) ChemRICH structural similarity enrichment analysis of metabolic perturbations detected in SDHC KO cells relative to control cells. Colors indicate mean log 2 (fold change) for the respective metabolite structural classes. (B) Changes in acyl-carnitine metabolites detected in SDHC KO cells relative to control cells (* denotes p-value < 0.05 by two-sided heteroscedastic t-test, adjusted by FDR for multiple comparisons). Bar color denotes acyl chain length [red: short (< 7), green: medium (7 - 12), blue: long (> 12)]. (C) Changes in adenine nucleotides detected in SDHC KO cells relative to control cells. Statistical analysis was as in (B). (D) Changes in NAD + biosynthetic precursors detected in SDHC KO cells relative to control cells. Statistical analysis was as in (B). (E) Quantification of relative sirtuin activity in SDHC KO and control cells by flow cytometry using an acylation-dependent, genetically-encoded fluorescent probe (* denotes p-value < 0.05 by Wilcox rank-sum test). (F) SILAC proteomics aggregate site-specific quantification ratios of acylated peptides normalized by corresponding protein abundance in SDHC KO and control cells. (G) Western blot quantification of H3K27ac normalized to total H3 (* denotes p-value < 0.05 by two-sided t-test). (H) Western blot quantification of H4ac (K5, K8, K12, and K16) normalized to total H4 (* denotes p-value < 0.05 by two-sided t-test). (I) Western blot quantification of pan-propionyl H3 normalized to total H3. (J) Western blot quantification of pan-butyryl H3 normalized to total H3 (* denotes p-value < 0.05 by two-sided t-test). (K) Western blot quantification of pan-malonyl H3 normalized to total H3. (L) Western blot quantification of pan-succinyl H3 normalized to total H3 (* denotes p-value < 0.05 by two-sided t-test). (M) Schematic illustration of metabolic effects observed in SDHC KO cells contributing to the chromatin hyperacylation phenotype. Error bars indicate standard deviations in all cases.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Control, Activity Assay, Flow Cytometry, Multiplex sample analysis, Quantitative Proteomics, Western Blot

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: (A) SeqMonk genomic view of epigenomic profiles generated from CUT&RUN and ChIP-seq at 5 kbp resolution for control cell line. Error bars indicate STDEV for CPM-normalized probe quantities from biological replicate experiments. (B-H) Fragment length histograms from CUT&RUN profiling in SDHC KO and control cell lines using antibodies specific to H3K27ac, H3K4me3, H3K27me2, CTCF, acetyllysine, propionyllysine, and butyryllysine. (I) ChromHMM emission parameter correlations plotted as a function of number of states in stacked epigenomic model. (J) ChromHMM emission parameters. (K) ChromHMM genomic enrichments for RefSeq annotations. (L) ChromHMM TSS enrichment patterns. (M) ChromHMM TES enrichment patterns. (N) ChromHMM state descriptions. (O) Boxplot of ATAC-seq coverage for ChromHMM states.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Generated, ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: NucleoATAC analysis of nucleosome position and fuzziness (delocalization) was performed for ChromHMM-called chromatin states. X-axes denote parameter fold-change and y-axes denote –log 10 (p-value) calculated by Wilcox rank-sum test. (A) Analysis of nucleosome inter-dyad distance changes in SDHC KO cells relative to control. (B) Analysis of nucleosome fuzziness changes in SDHC KO cells relative to control. (C) Analysis of nucleosome inter-dyad distance changes in HATi-treated [MB-3 (100 µM) and C646 (10 µM) for 72 h] SDHC KO cells relative to vehicle-treated SDHC KO cells. (D) Analysis of nucleosome fuzziness changes in HATi-treated [MB-3 (100 µM) and C646 (10 µM) for 72 h] SDHC KO cells relative to vehicle-treated SDHC KO cells.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Control

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: (A) Analysis of fragment length from ATAC-seq experiments in SDHC KO and control lines. (B) ATAC-seq peak numbers for SDHC KO and control lines identified by the MACS peak-calling algorithm. (C) Analysis of genomic localization of ATAC-seq peaks in SDHC KO and control cells. (D) Western blot analysis of H3K27ac and total H3 in SDHC KO cells treated with HATi (10 µM C646, 100 µM MB-3) for 24-72 h. (E) Quantification of relative H3K27ac/H3 ratio as a function of HATi treatment duration. * denotes two-tailed p-value < 0.05 by unpaired t-test. (F) Analysis of fragment length from ATAC-seq experiments in SDHC KO cells either treated with HATi (10 µM C646, 100 µM MB-3) or vehicle for 72 h. (G) ATAC-seq peak numbers for SDHC KO cells either treated with HATi (10 µM C646, 100 µM MB-3) or vehicle for 72 h identified by the MACS peak-calling algorithm. (H) Analysis of genomic localization of ATAC-seq peaks in SDHC KO cells either treated with HATi (10 µM C646, 100 µM MB-3) or vehicle for 72 h.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Control, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: (A,B) Flow cytometry analysis of cell cycle stages in SDHC KO and control cells by DAPI-Geminin staining. (C) Insulation scores calculated for first 20 Mbp of chromosome 1 for biological replicate experiments for SDHC KO and control cells. (D) Heatmap showing genome-wide correlation for insulation between insulation scores calculated for biological replicate SDHC KO and control cells. (E,F) Analysis of initial cellular fluorescence for ROIs in FRAP experiments using mEmerald-STAG2 (E) and mEmerald-RAD21 (F). Statistical comparison of distributions is performed using a Wilcox rank-sum test. (G) Quantifications of change in expression of CTCF (black), cohesin subunits (red) and known cohesin regulators (blue) in SDHC KO relative to control cells by SILAC proteomics. (H,I) Genome-wide CTCF footprints calculated from ATAC-seq data for SDHC KO and control cells. (J) Quantification of CpG methylation at CTCF binding sites in SDHC KO and control cells (“ns” denotes lack of statistical significance in comparison of distributions by Wilcox rank-sum test). (K) Western blot analysis of SMC3ac Lys105/106 and total SMC3 in SDHC KO and control cell lines. (L) Quantitative analysis of SMC3ac Lys 105/106 relative to total SMC3. Statistical comparison by unpaired two-tailed t-test.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Flow Cytometry, Control, Staining, Insulation, Genome Wide, Fluorescence, Comparison, Expressing, Multiplex sample analysis, CpG Methylation Assay, Binding Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: (A) HiCRep stratum-adjusted correlation coefficients calculated at 1 Mbp resolution for biological replicate eHi-C datasets. (B) TAD boundary strengths in SDHC KO and control cells (* denotes p-value < 0.05 by Wilcox rank-sum test). (C) KR-normalized eHi-C contact matrices for SDHC KO (x-axis) and control (y-axis) eH-C datasets showing chromosome 3 (0-140 Mbp) at 250 Kbp resolution. (D) Subset of (C) showing SDHC KO (x-axis) and control (y-axis) eH-C chromosome 4 (69-82 Mbp) at 25 Kbp resolution. Arrows indicate positions of identified TAD boundaries. Arrows with stems indicate increased boundary strength in SDHC KO cells relative to control [log 2 (fold-change) > 1]. Arrowheads without stems indicate TADs with unchanged boundary strength. (E) Plot of TAD boundary strength in SDHC KO and control cells. Colors indicate TADs with increased [log 2 (fold-change) > 1[and decreased [log 2 (fold-change) < −1] boundary strength in SDHC KO relative to control cells. (F) Aggregate normalized FRAP curves for mEmerald-STAG2 transiently expressed in SDHC KO (N=12) and control (N=13) cells. Ranges indicate SEM. (G) Aggregate normalized FRAP curves for mEmerald-RAD21 transiently expressed in SDHC KO (N=24) and control (N=15) cells. Ranges indicate SEM. (H) Acyl PTMs identified on cohesin subunits via SILAC proteomics. Amino acid positions on respective cohesin subunits are indicated. Colors indicate PTM presence (gray: unchanged SDHC KO vs. control; black: uniquely identified in SDHC KO). (I) Estimated protein-normalized acyl PTM SILAC H/L ratios. For PTMs only identified in SDHC KO line, H/L ratio estimated as 1/(S/N ratio). (J) Venn diagram of acetylation and non-acetyl acylation site locations. (K) Histogram of number of PTM types per site identified.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Control, Multiplex sample analysis

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: (A) Pearson correlation coefficient matrix calculated from eHi-C contact matrices of SDHC KO (x-axis) and control (y-axis) chromosome 4 (0-155 Mb) at 500 Kbp resolution. Compartment eigenvectors, ChromHMM annotations, and ATAC-seq peaks are shown. (B) Genome-wide compartment autocorrelations calculated as a function of bin separation along the linear genome for SDHC KO and control cells. (C) Compartment autocorrelations within “A” compartments. (D) Compartment autocorrelations within “B” compartments. (E) Calculated differences in compartment autocorrelation between SDHC KO and control cells. (F-H) Boxplots of CPM-normalized reads per 250 Kbp bin for genomic regions mapping to A and B compartments with conserved identities in SDHC KO and control cells for (F) H3K27ac CUT&RUN, (G) H3K4me3 CUT&RUN, and (H) H3K27me3 ChIP-seq. (I) Boxplot of RNA-seq TPM-normalized expression quantities for transcripts mapping to A and B compartments in control cells. (J) Analysis of RNA-seq TPM-normalized expression changes for genes mapping to compartments that convert between A/B in SDHC KO cells relative to genes mapping to compartments that do not convert. (K) Representative images from multiplexed staining and volumetric imaging of fixed cell nuclei. Voxel dimension is 140 nm square horizontally and 320 nm vertically. (L) Representative segmented nuclear model derived from DAPI 3D image stack using Trainable Weka Segmentation. (M) Western blot of H3K27ac and total H3 for control cells treated with oligomycin (1 µM for 24 h), oligomycin and C646/MB-3 (10 µM C646, 100 µM MB-3 for 24 h), or EX-527 (20 µM for 24 h). (N) Analysis of H3K27ac intensity ratio for DAPI-stained puncta relative to non-puncta regions of nucleus. (O) Analysis of H3K27me3 intensity ratio for DAPI-stained puncta relative to non-puncta regions of nucleus. (P) Quantification of DAPI puncta total volume per cell. (Q) Quantification of DAPI puncta percentage of nucleus volume. * denotes p-value < 0.05 by Wilcox rank-sum test.

Article Snippet: Antibodies used in this analysis include anti-SDHC rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, cat# sc-67256 (M-169), 1:500 dilution), anti-actin rabbit polyclonal IgG antibody (Sigma, cat# A2066, 1:500 dilution), anti-SIRT1 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 9475, 1:1000 dilution), anti-Phospho SIRT1 (Ser47) rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 2314, 1:2000 dilution), anti-SIRT2 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12650, 1:1000 dilution), anti-SIRT3 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5490, 1:1000 dilution), anti-SIRT5 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 8782, 1:1000 dilution), anti-SIRT6 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 12486, 1:1000 dilution), anti-SIRT7 rabbit monoclonal IgG antibody (Cell Signaling Technology cat# 5360, 1:1000 dilution), anti-histone H3K27ac rabbit polyclonal IgG antibody (Novus Biologicals cat# NBP2-54615, 1:1000 dilution), anti-histone H4ac (H4K5ac, H4K8ac, H4K12ac, and H4K16ac) rabbit monoclonal IgG antibody (Abcam cat# ab177790, 1:20000 dilution), pan anti-propionyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-201), pan anti-butyryl rabbit polyclonal antibody (PTM Biolabs cat# PTM-301), pan anti-malonyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-901), pan anti-succinyl rabbit polyclonal antibody (PTM Biolabs cat# PTM-401), anti-histone H4 rabbit polyclonal IgG antibody (Abcam cat# ab7311, 1:1000 dilution), and anti-histone H3 rabbit polyclonal IgG antibody (Abcam cat# ab1791, 1:1000 dilution).

Techniques: Control, Genome Wide, ChIP-sequencing, RNA Sequencing, Expressing, Staining, Imaging, Derivative Assay, Western Blot