Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: PARP1, Ku80, and Ku70 are dramatically elevated in patients with aggressive HBL and bind to the core sequence of ALCDs. a The core 18BPS was linked to biotin beads and was incubated with nuclear extracts isolated from HBL with low and high levels of TSPs. Coomassie staining identified four proteins (shown on the right) that specifically interacted with the 18BPS in nuclear extracts from aggressive HBL. b Large-scale isolation and mass spec analysis of the interacting proteins. The proteins which are specifically interacting with 18BPS are PARP1, Ku80, Ku70, and several additional proteins, some of them belong to nuclear matrix (Supplementary Fig. ). c , d Expression of PARP1, Ku80, and Ku70 is dramatically increased in patients with aggressive HBL. QRT-PCR ( c ) and western blotting ( d ) were performed with mRNAs and proteins isolated from HBL samples with low and high TSPs levels. SHP is a small heterodimer partner, which was detected by re-probe of the PARP1 membrane and which serves as a good control for protein loading. e PARP1, Ku80, and Ku70 form a complex in livers of patients with aggressive HBL. Ku70 and Ku80 were precipitated and PARP1 and Ku70 or Ku80 were determined in these IPs. IgG: heavy chains of IgG. f Fractionation of nuclear proteins from background (blue) and HBL (black) sections of aggressive HBLs by HPLC-based size exclusion chromatography (SEC). Red arrow shows elevation of optical density in the area of high MW protein–protein complexes. g Top: Examination of PARP1/Ku80/Ku70 complexes in SEC fractions. Western blotting shows the amount of proteins in each fraction. Bottom: PARP1-IP shows immunoprecipitation of PARP1 and analysis of Ku70 in these IPs. Images below show hypothetical compositions of PARP1 complexes. Error bars represent standard error of the mean ( c )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Sequencing, Incubation, Isolation, Staining, Mass Spectrometry, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Control, Fractionation, Size-exclusion Chromatography, Immunoprecipitation

Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: ALCDs are activated in aggressive HBL samples and in hepatoblastoma cancer cells by PARP1/Ku80/Ku70 complexes. a ChIP analysis of the ten representative ALCDs and two negative controls, which contain the inactive 250 bp domain (shown on the left and right) in background and HBL sections of the livers. M marker, In input, B beads, PP1 PARP1, H3K9-Ac histone H3 acetylated at K9, 3-me histone H3 trimethylated at K9. b Quantitative presentation of ChIP analysis. Amounts of PARP1/Ku80/Ku70 complexes were calculated as average of PARP1, Ku80, and Ku70 signals and then as percentage of this average signal to input. c ChIP analysis of ten representative ALCDs in HepG2 cells with and without treatment with 100 µM DPQ. d Quantitative presentations of ChIP analysis. Calculations were performed as described above. e Cell proliferation assay in HepG2, Huh6, and B6-2 cells treated with 100 µM DPQ for 72 h. Representative images highlighting changes in cell proliferation after DPQ treatment are shown in Supplementary Fig. . f Inhibition of PARP1 by DPQ reduces PARP1/Ku80/Ku70 complexes in HeLa and in HepG2 cells and reduces expression of TSPs and cell cycle proteins. Cells were treated with DPQ for 24 and 48 h and western blotting and Co-IPs were performed as described above. Error bars represent standard error of the mean ( e )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Marker, Proliferation Assay, Inhibition, Expressing, Western Blot

Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: Inhibition of PARP1 by FDA-approved inhibitor DPQ and by si-PARP1 siRNA eliminates PARP1 complexes leading to silence of ALCDs and to inhibition of cell proliferation. a Western blotting of proteins isolated from Huh6 cells treated with 100 µM DPQ for 48 h. Ku80-IP: Ku80 was immunoprecipitated and PARP1 was examined in these IPs. Bar graphs below show levels of proteins as ratios to β-actin. b HepG2 and Huh6 cells were transfected with siRNA-targeting PARP1. Expression of proteins shown on the left and right was analyzed by western blotting. Ku80-IP: western shows the disruption of PARP1/Ku80/Ku70 complexes by inhibition of PARP1. c Cell proliferation assay of HepG2, Huh6, and B6-2 cells transfected with si-PARP1. The assay was performed 48 h after transfection of siRNA. d Representative images highlighting changes in cell proliferation with si-PARP1 transfection in B6-2 cells. e Scratch assay of proliferation of HepG2 cells untreated (con) and treated with DPQ. Percentage shows % of not-closed scratches (inhibition of proliferation) at 48 h after scratch. f Cell proliferation assay shows inhibition of proliferation of HepG2 cells by low concentrations of DPQ and olaparib (Ola). Right image shows Co-IP of PARP1/Ku80/70 complexes and western blotting of downstream targets of ALCDs. g ChIP assay of the ALCD regions of within C/EBPα, HACE1, p53, and β-catenin genes in control and olaparib-treated HepG2 cells. Experiment was performed as described in legend to Fig. . Bottom images show quantitation of ChIP for ALCDs in these genes. h A diagram showing hypothesis which is based on the results of these studies. Aggressive liver cancer is associated with elevation of PARP1, which forms complexes with Ku80 and Ku70 and subsequent chromatin remodeling around ALCDs that leads to a dramatic activation of multiple pathways of liver cancer. An important part of this hypothesis is that certain TSPs are also activated by PARP1-ALCDs axis, but they are posttranslationally modified and are converted into proteins with potential oncogenic activities. Oncogenic activities of posttranslationally modified C/EBPα are shown in our recent publication . Error bars represent standard error of the mean ( c , f )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Inhibition, Western Blot, Isolation, Immunoprecipitation, Transfection, Expressing, Disruption, Proliferation Assay, Wound Healing Assay, Co-Immunoprecipitation Assay, Control, Quantitation Assay, Activation Assay, Modification

Journal: Human Reproduction (Oxford, England)

Article Title: The role of small extracellular vesicle-miRNAs in endometriosis

doi: 10.1093/humrep/dead216

Figure Lengend Snippet: Studies investigating the differential expression of sEV-miRNA.

Article Snippet: 13 , Uterine aspirate fluid , Human samples: Endometriosis (stage III–IV, endometrioma, diagnosed via laparoscopy), n=22 Control (including simple ovarian cyst, uterine leiomyoma, tubal ligation), n=25 *Mentioned about that findings were regardless of secretory or proliferative phases in the results section but did not declare number of samples that were of proliferative or secretory. , Size exclusion chromatography exosome isolation kit (Echobiotech, Beijing, China) , NTA : Median size: 111.7 nm and the proportion of the main peak was 94.1%. Visualised via TEM . , WB : CD63, TSG101, and HSP70 positive. Calnexin negative. , Microarray analysis of miRNA from sEVs and endometrial tissues from uterine aspirate fluid, followed by qRT-PCR . sEV uptake experiments with JNK activator anisomycin by M0 macrophages followed by flow cytometry , WB, and MAPK phosphorylation antibody array . Transwell migration and invasion assay : Co-culture of endometrial tissues of control group and sEV-treated macrophages. Transfection of M0 macrophages with hsa-miR-210-3p mimic/inhibitor/corresponding NC followed by qRT-PCR and flow cytometry . , ↑ hsa-miR-210-3p Regardless of secretory or proliferative phases , Jiang et al. (2022) .

Techniques: Expressing, Isolation, Concentration Assay, Western Blot, Microarray, Quantitative RT-PCR, Mouse Assay, Immunohistochemistry, Extraction, Transfection, Transmission Electron Microscopy, Sequencing, Flow Cytometry, Reporter Gene Assay, Zeta Potential Analyzer, Cell Culture, In Situ, Hybridization, Wound Healing Assay, Marker, Ligation, Migration, Size-exclusion Chromatography, Ab Array, Invasion Assay

Journal: STAR Protocols

Article Title: Protocol for generating and characterizing a nasal epithelial model using imaging with application for respiratory viruses

doi: 10.1016/j.xpro.2024.103520

Figure Lengend Snippet: ACE2 and TMPRSS2 mRNA expression over the course of epithelium maturation Cells were collected prior to ALI (D0), and at 21 (D21) and 42 (D42) days post-air-liquid interface (ALI) and analyzed for (A) ACE2 and (B) TMPRSS2 mRNA expression ( n = 4 tissue donors). Triplicate wells were averaged for each tissue donor shown as dots; lines show mean Ct corrected for RPLP0 ± SEM. Friedman’s test and Wilcoxon matched pairs test were used to test for significance (ns: not significant). ACE2 mRNA expression increased significantly from D0 undifferentiated cells to D42 mtured epithelia (p = 0.04). Similarly, TMPRSS2 mRNA expression increased significantly in matured epithelia at D42 compared to D0 (p = 0.014).

Article Snippet: ∗TaqMan Gene Expression Assay, VIC primer-limited Gene: Rplp0, assay ID: Hs00420895_gH, dye: VIC-MGB_PL, , Applied Biosystems , Cat# 4448484.

Techniques: Expressing

Journal: STAR Protocols

Article Title: Protocol for generating and characterizing a nasal epithelial model using imaging with application for respiratory viruses

doi: 10.1016/j.xpro.2024.103520

Figure Lengend Snippet: Mature nasal epithelial cells support SARS-CoV-2 Omicron BA.1 infection Epithelia prior to air-liquid interface (D0), 21 (D21) and 42 (D42) days post air-liquid interface (ALI) were infected at 0.01 MOI. Cell-associated viral N gene mRNA normalized to RPLP0 was quantified using RT-qPCR ( n = 4 tissue donors). Triplicate wells were collected and averaged for each tissue donor. Friedman’s test and Wilcoxon signed rank test were used to test for significance (ns: not significant). There was a significant increase in intracellular SARS-CoV-2 viral load in matured epithelia at D42 compared to undifferentiated cells at D0 (p = 0.014). Lines represent means ± SEM.

Article Snippet: ∗TaqMan Gene Expression Assay, VIC primer-limited Gene: Rplp0, assay ID: Hs00420895_gH, dye: VIC-MGB_PL, , Applied Biosystems , Cat# 4448484.

Techniques: Infection, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Protocol for generating and characterizing a nasal epithelial model using imaging with application for respiratory viruses

doi: 10.1016/j.xpro.2024.103520

Figure Lengend Snippet:

Article Snippet: ∗TaqMan Gene Expression Assay, VIC primer-limited Gene: Rplp0, assay ID: Hs00420895_gH, dye: VIC-MGB_PL, , Applied Biosystems , Cat# 4448484.

Techniques: Virus, Control, Recombinant, Staining, Electron Microscopy, Sterility, Enzyme-linked Immunosorbent Assay, Gene Expression, Software, Microscopy, Membrane, Cell Culture, Pore Size, Marker, Adhesive, Low Protein Binding, Fluorescence, Inverted Microscopy

Journal: STAR Protocols

Article Title: Protocol for generating and characterizing a nasal epithelial model using imaging with application for respiratory viruses

doi: 10.1016/j.xpro.2024.103520

Figure Lengend Snippet: Luna Universal Probe Master Mix

Article Snippet: ∗TaqMan Gene Expression Assay, VIC primer-limited Gene: Rplp0, assay ID: Hs00420895_gH, dye: VIC-MGB_PL, , Applied Biosystems , Cat# 4448484.

Techniques: Gene Assay

Journal: STAR Protocols

Article Title: Protocol for generating and characterizing a nasal epithelial model using imaging with application for respiratory viruses

doi: 10.1016/j.xpro.2024.103520

Figure Lengend Snippet: TaqMan Fast Virus 1-Step Master Mix

Article Snippet: ∗TaqMan Gene Expression Assay, VIC primer-limited Gene: Rplp0, assay ID: Hs00420895_gH, dye: VIC-MGB_PL, , Applied Biosystems , Cat# 4448484.

Techniques: Virus

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 initially delays Delta16HER2-driven tumorigenesis, but then promotes a high tumor multiplicity. ( A ) Breeding scheme used to obtain Delta16HER2/SIRT6-OE female mice and a representative genotyping PCR. ( B ) Kaplan-Meir curves comparing the percentage of tumor-free mice between Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 17/group). ( C ) Representative post mortem pictures of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age (top panels), and relative H&E staining of the primary mammary tumor masses (bottom panels). Black arrows indicate tumor foci. ( D , E ) Tumor multiplicity and tumor growth curves of Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 17/group). ( F ) mRNA expression of Sirtuin1-7(Sirt1-7) and Delta16HER2 normalized to β-Actin mRNA level in tumors of 30-week-old Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 4/group). ( G ) Representative images of IHC staining for SIRT6 (brown) in tumors of 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice, respectively. Delta16HER2/SIRT6-OE tumors show a strong SIRT6 level and mainly localized into the nucleus. Scale bar, 25 mm. ( H , I ) Western blot analysis and relative quantification of total SIRT6 and phosphorylated SIRT6 (pSIRT6-Ser388) normalized to β-Actin protein level. pSIRT6-Ser388/total SIRT6 represents the ratio of β-Actin-normalized phosphorylated protein over the total SIRT6 protein. ( J ) Flow cytometry staining for Delta16HER2 and SIRT6 of primary cells (passage 4 in vitro) derived from tumors of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age. MFI, Median Fluorescence Intensity. The experiment was carried out in triplicates. In ( B ) ***p = 0.0008 (Log-rank test); in ( D , E ) **p < 0.01, ***p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparisons test); in ( F , H , J ) *p < 0.05, **p < 0.01 (two-tailed unpaired t test). Error bars represent SD. See also Fig. .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Quantitative Proteomics, Flow Cytometry, In Vitro, Derivative Assay, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 boosts tumor cell migration, invasion and lung metastasis in Delta16HER2/SIRT6-OE mice. ( A ) Representative pictures of H&E staining on lungs of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age (n = 7/group). Dashed black lines highlight the metastatic area in each picture. Quantification is represented as number of metastasis per mouse ( B ) and as metastasis area over total section area ( C ). Both number and area of metastasis were calculated as average over 2–6 consecutive tissue sections per mouse. ( D ) Soft agar assay performed on primary cells (passage 4 in vitro) derived from tumors of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age. Stereomicroscope images, 4X magnification (top) and 10X zoom (bottom). Number of colonies ( E ) were quantified using ImageJ software. Results are expressed as % of total number of seeded cells. The assay was performed in triplicates. ( F , G ) Transwell migration assay and quantification of Delta16HER2 and Delta16HER2/SIRT6-OE primary cells. Pore size 8.0 mm. Quantification is reported as percentage of migrating cells over the total number of seeded cells. In ( B , C , E , G ) *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t test). Error bars represent SD.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Migration, Staining, Soft Agar Assay, In Vitro, Derivative Assay, Software, Transwell Migration Assay, Pore Size, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 prevents tumor cells from Delta16HER2-induced G2/M arrest and senescence. ( A ) Ex vivo cell cycle analysis of cell suspensions derived from Delta16HER2 and Delta16HER2/SIRT6-OE tumors at 20 weeks of age (top panels) and at 30 weeks of age (bottom panels). DNA content was evaluated by flow cytometry using propidium iodide (PI) staining (n = 3) and then analyzed using Dean-Jett-Fox algorithm in FlowJo software. Percentages of cells in each cell cycle phase are summarized in each panel. ( B ) Immunoblot image and quantification of Cyclin D1 and Cyclin E levels normalized to β-Actin in tumors of either 20- (top) or 30-week-old (bottom) Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4). ( C ) mRNA expression of Trp53 , Cdkn2a and Cdkn1a genes normalized to β-Actin mRNA level in tumors of 30-week-old Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 4/group). ( D ) Detection of senescence-associated β-galactosidase (SA-β-Gal) activity in frozen tumors of 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE (n = 3). Quantification is expressed as % of SA-β-Gal positive area (blue) with respect to the total section area. Scale bar, 25 mm. In ( B – D ) ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t test). Error bars represent SD. See also Fig. .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Ex Vivo, Cell Cycle Assay, Derivative Assay, Flow Cytometry, Staining, Software, Western Blot, Expressing, Activity Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 promotes stemness and self-renewal capacity of Delta16HER2 tumor cells. ( A – D ) Flow Cytometry analysis of CD44, CD24, OCT3/4, Notch1 and NANOG levels in primary tumor cells derived from 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4). Quantification is expressed as median fluorescence intensity (MFI) ( A ) and as percentage of positive cells ( C and D ). ( E ) Representative images of mammosphere formation assay performed on primary tumor cells derived from 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4, top panel). Quantification is represented for 2 serial cloning assays and is expressed as percentage of mammosphere forming units (MFU) with respect to the total number of seeded cells (bottom panel). See also Fig. . ( F ) Flow cytometry analysis of SIRT6 and HER2 expression in CAM6 cells stably transduced with pLENTI-Blank (empty vector) and pLENTI-SIRT6. ( G ) Mammosphere formation assay on CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6. Scale bar, 200 mm. ( H , I ) Quantification of mammosphere forming capacity (MFU %) and average sphere area of CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6 cells. Experiment was carried out in triplicate. ( J ) Immunofluorescence staining of OCT3/4 on CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6 mammospheres. DAPI is used for nuclei counterstaining. Magnification 40X. In ( A , C – E , H , I ) *p < 0.05, **p < 0.01, ***P < 0.001 (two-tailed unpaired t test). Error bars represent SEM.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Flow Cytometry, Derivative Assay, Fluorescence, Tube Formation Assay, Cloning, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Immunofluorescence, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 is amplified in a subset of HER2-positive breast cancer patients and correlates with poor relapse-free-survival. ( A ) cBioPortal data summarizing the type and frequency of alteration of HER2 ( ERBB2 ) and SIRT6 genes in sequenced samples from publicly available invasive breast cancer studies (listed in the “study of origin” section). A total of 4860 and 4379 breast cancer patients were profiled for ERBB2 and SIRT6 alterations, respectively. ( B ) Co-occurrence/Mutual exclusivity analysis from cBioPortal indicating the number of patient samples that have amplification of HER2 (ERBB2) and SIRT6 alone, in both or neither genes. Odd ratio = neither*both/SIRT6 not ERBB2*ERBB2 not SIRT6. Log2 Odd Ratio > 1 indicates co-occurrence of HER2 and SIRT6 amplifications (p < 0.0001, Pearson correlation). ( C – E ) Kaplan–Meier plots from GOBO database using overall survival ( C ) and relapse-free survival (RFS) ( D , E ) as outcome in all HER2-enriched breast cancer ( D ) and in grade 3 HER2-enriched breast cancer ( E ). Data have been stratified into the two quantiles based on SIRT6 gene expression level (SIRT6_low, grey line and SIRT6_high, red line) using 10-year censoring as endpoint. ( F ) DNA microarray data from bc-GenExMiner showing SIRT6 expression in HER2- (n = 4068) and HER2 + (n = 680) breast cancer samples. ( G , H ) Kaplan–Meier plots from bc-GenExMiner using distant metastasis free survival (DMFS) and disease free survival (DFS) as outcomes in HER2-enriched breast cancer. Data have been stratified into the two quantiles based on SIRT6 gene expression level (SIRT6_low, purple line and SURT6_high, blue line). HR (hazard ratio) and 95% CI (confidence interval) are reported in the figure. In ( C , D , F , G ) *p = 0.02436, *p = 0.02143, *p = 0.0468, *p = 0.0222 (Log-rank test). In ( E ) *p = 0.0322 (Welch’s test). See also Figs. and .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Amplification, Gene Expression, Microarray, Expressing

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: ChIP- and RNA-seq identify TBX3 as a SIRT6 target and a prognostic marker in HER2 + breast cancer. ( A ) Venn diagram representing the genes that are differentially expressed and differentially bound by H3K9ac (cutoff FDR ≤ 0.05) in tumors from Delta16HER2/SIRT6-OE versus Delta16HER2 controls (20 weeks of age, n = 2 for RNA-seq and n = 2–3 for ChIP-seq). ( B , C ) ChIP and RNA-seq tracks showing H3K9ac level and expression for Il12a and Tbx3 genes in Delta16HER2 (blue) versus Delta16HER2/SIRT6-OE (red) tumors (at 20 weeks of age). ( D ) Bee swarm plots computed in bc-GenExMiner showing the expression levels of IL12A and TBX3 in HER2–/ + breast cancers. ( E , F ) Kaplan–Meier plots from bc-GenExMiner using distant metastasis free survival (DMFS) and disease free survival (DFS) as outcomes in HER2-enriched breast cancer. Data have been stratified into the two quantiles based on IL12A gene expression level (( E ), IL12A_low, purple line and IL12A_high, blue line) and TBX3 gene expression level (( F ), TBX3_low, purple line and TBX3_high, blue line). HR (hazard ratio) and 95% CI (confidence interval) are reported in the figure. In ( D ) ****P < 0.0001 (Welch’s test). In ( E , F ) ns, p = 0.1124; ns, p = 0.7269; ***p = 0.0004, **p = 0.0075 (Log-rank test). See also Figs. and .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: RNA Sequencing, Marker, ChIP-sequencing, Expressing, Gene Expression

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6-OE induces loss of TBX3 and aggressiveness in both mouse and human models of Delta16HER2/HER2 + . ( A ) Representative pictures and ( B ) quantification for Tbx3 IHC in the indicated mouse tumors. ( C ) Immunoblot of Tbx3 in mouse tumors of the indicated genotype (20 weeks, n = 4 per group). ( D ) Representative pictures of the human BT474 cell line transfected with either pHIV-dTomato or pHIV-SIRT6-dTomato. ( E ) Real-time PCR for SIRT6 and TBX3 expression in sorted BT474 transfected as indicated. Not transfected HEK293 were used as control sample and TBP as housekeeping gene. ( F ) Representative pictures and ( G ) quantification of the Transwell migration assay of the human BT474 cell line transfected with either pHIV-dTomato or pHIV-SIRT6-dTomato. ( H ) Representative pictures and ( I ) number and area quantification of pHIV-dTomato and pHIV-SIRT6-dTomato BT474 mammospheres. MFU% = % of mammary forming units over total number of seeded cells. A.U . arbitrary unit. Bars indicate mean ± SD. In ( B ) ***p = 0.0009; ( C ) **p = 0.0077; ( D ) **p = 0.0014, ***p = 0.0003; ( G ) **p = 0.0077 and ( I ) statistics indicates unpaired two-tailed t test.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, Control, Transwell Migration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: Loss of TBX3 mimics SIRT6 -OE in vitro and predicts poor survival of HER2-positive breast cancer patients. ( A ) Immunoblot and quantification of TBX3 and GAPDH in lysates of BT474 cell line transfected with the indicated siRNA (48 h post transfection, n = 2 independent replicates). ( B ) Representative pictures and ( C ) quantification of the Transwell migration assay of the human BT474 cell line transfected as indicated (n = 5). ( D ) Representative pictures and ( E ) number and area quantification of siRNA control and siRNA TBX3 BT474 mammospheres (n = 3). MFU% = % of mammary forming units over total number of seeded cells. A.U . arbitrary unit. Bars indicate mean ± SD. See also Fig. . ( F ) Lollipop graph from cBioPortal showing mutations of the TBX3 gene in breast cancer patients (n = 338). Y axis represents the number of mutations while X axis indicates the correspondent amino acid position. OncoKB tracks in blue indicate those mutations that are likely-oncogenic and likely loss-of-function. Types of mutation are color-coded in the figure legend. ( G ) Summary of total number and frequency of TBX3 mutations in all, HER2-WT and HER2 amplified breast cancer subsets. Pie charts indicate the frequency of the different types of TBX3 mutations over total number of TBX3 mutations in each subset. ( H ) Kaplan–Meier plots from cBioPortal showing the overall survival of patients with HER2 amplified alone (n = 271), and those with concomitant SIRT6 amplification (n = 8) or TBX3 loss-of-function mutations (n = 20) . P = 0.0007 (Log-rank Mantel-Cox test). In ( A ) **p = 0.0047, ( C ) *p = 0.0282, ( E ) **p = 0.0051 and ns, p = 0.2106 indicates unpaired two-tailed t test.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: In Vitro, Western Blot, Transfection, Transwell Migration Assay, Control, Mutagenesis, Amplification, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: qRT-PCR primer list.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Sequencing, Amplification

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: Summary of used antibodies.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques:

Journal: International Journal of Nanomedicine

Article Title: M2 Macrophage-Derived Exosomal lncRNA MIR4435-2HG Promotes Progression of Infantile Hemangiomas by Targeting HNRNPA1

doi: 10.2147/ijn.s435132

Figure Lengend Snippet: Figure 1 Isolation and identification of M2-exos. (a). Representative pictures of THP-1 cells, M0 macrophages, and M2 polarized macrophages; (b). qRT-PCR analysis of expression of M0 polarized macrophage markers CD11b, CD14, and CD68; (c). qRT-PCR analysis of expression of M1 polarized macrophage marker iNOS and M2 polarized macrophage marker, IL-10, and CD206 -PCR analysis; (d). TEM observation of M2 polarized macrophage exosomes morphology (scale bar, 100 nm); (e). Nanoparticle particle size analysis showed that extracted exosomes were approximately 77.48 nm in diameter; (f). Western blotting detection of the expression of calnexin, HSP70, TSG101, and CD9 in exosomes of M0 polarized macrophages and M2 polarized macrophages and cell lysates; (g). Fluorescence microscopy of HemECs uptake of M2 exo (EvLINK labeled exosomes, CellLINK labeled cell membranes) (Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001; ns, nonsignificant).

Article Snippet: GB11618; Servicebio), CD9 (25 kDa; Rabbit; 1:2000; 20597-1-AP; Proteintech), α-tubulin (55 kDa; Rabbit; 1:3000; GB15201; Servicebio), HNRNPA1 (34 kDa; Rabbit; 1:10000; 11176-1-AP; Proteintech), NF-κB p65 (65 kDa; Mouse; 1:2000; 66535-1-lg; Proteintech), phospho-NF-κB p65 (65 kDa; Rabbit; 1:500; GB113882; Servicebio), IkBα (36 kDa; Mouse; 1:10000; 66418-1-lg; Proteintech), and IKB alpha (phospho S36) (35 kDa; Rabbit; 1:10000; ab133462; Abcam).

Techniques: Isolation, Quantitative RT-PCR, Expressing, Marker, Particle Size Analysis, Western Blot, Fluorescence, Microscopy, Labeling

Journal: PLoS ONE

Article Title: Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18

doi: 10.1371/journal.pone.0229672

Figure Lengend Snippet: A–SDS-PAGE gel of MBP-trap purification of pMal MBP-18E6. Lane 1 designated the molecular weight marker, lane 2 is protein loaded on the column, lane 3 is the flow-through from the column, lanes 4 and 5 are independent collections of the peak of the primary elution, lane 6 is empty, lanes 7–9 is raw load protein (7), column flow-through (8), and peak of the primary elution (9) subjected to DTT reduction. B–SDS-PAGE gel of MBP-HPV18 E6 protein prior to and following DTT reduction with a Hi trap Q anion exchange column. Lane 1 designated the molecular weight marker, lane 2 is protein loaded on the column, lane 3 is the flow-through from the column, lanes 4 and 5 are independent collections of the peak of the primary elution, lanes 6–8 is raw load protein (6), column flow-through (7), and peak of the primary elution (8) subjected to DTT reduction. C–Size-exclusion chromatography of the peak of the primary elution subjected to DTT reduction in Fig 1A. D–Mass spectrometry of MBP-HPV18 E6 protein from re-folded and reduced, peak eluate solution from the QHP column.

Article Snippet: Type-specific mouse monoclonal antibodies to HPV16/18 E6 (Clone C1P5, Santa Cruz Biotechnology, Santa Cruz, USA), HPV16 E7 (Clone 716–281, ThermoFisher Scientific, South San Francisco, USA), HPV18 E6 (Clone G-7, Santa Cruz Biotechnology, Santa Cruz, USA), and HPV18 E7 (Clone F-7, Santa Cruz Biotechnology, Santa Cruz, USA) were purchased for specificity experiments.

Techniques: SDS Page, Purification, Molecular Weight, Marker, Size-exclusion Chromatography, Mass Spectrometry

Journal: PLoS ONE

Article Title: Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18

doi: 10.1371/journal.pone.0229672

Figure Lengend Snippet: Concentration assignment of the pooled human donor reference serum from observed ECL signal.

Article Snippet: Type-specific mouse monoclonal antibodies to HPV16/18 E6 (Clone C1P5, Santa Cruz Biotechnology, Santa Cruz, USA), HPV16 E7 (Clone 716–281, ThermoFisher Scientific, South San Francisco, USA), HPV18 E6 (Clone G-7, Santa Cruz Biotechnology, Santa Cruz, USA), and HPV18 E7 (Clone F-7, Santa Cruz Biotechnology, Santa Cruz, USA) were purchased for specificity experiments.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18

doi: 10.1371/journal.pone.0229672

Figure Lengend Snippet: Group A–HPV+ cervical cancer subjects (n = 3); Group B–normal, healthy adult donors (n = 4); Group C–normal pediatric donors (n = 10). A–inter-subject variability for evaluating the concentration of IgG anti-HPV16 E6 over multiple assays (n = 5 assays). B–inter-subject variability for evaluating the concentration of IgG anti-HPV16 E7 over multiple assays (n = 6 assays). C—inter-subject variability for evaluating the concentration of IgG anti-HPV18 E6 over multiple assays (n = 5 assays). D—inter-subject variability for evaluating the concentration of IgG anti-HPV18 E7 over multiple assays (n = 5 assays). E–Inter-group anti-HPV IgG concentrations plotted as median ± interquartile range. Open shapes are Groups A and B and filled shapes is Group C.

Article Snippet: Type-specific mouse monoclonal antibodies to HPV16/18 E6 (Clone C1P5, Santa Cruz Biotechnology, Santa Cruz, USA), HPV16 E7 (Clone 716–281, ThermoFisher Scientific, South San Francisco, USA), HPV18 E6 (Clone G-7, Santa Cruz Biotechnology, Santa Cruz, USA), and HPV18 E7 (Clone F-7, Santa Cruz Biotechnology, Santa Cruz, USA) were purchased for specificity experiments.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18

doi: 10.1371/journal.pone.0229672

Figure Lengend Snippet: Serum from HPV+ cervical cancer subjects (n = 3), and normal, healthy adult donors (n = 4) was evaluated to identify the linear range for sample testing over 2 assays. Concentration for each sample was normalized to the observed concentration at 800-fold and plotted as a percent change from this value across 8 dilutions. A–dilutional linearity for seven samples in relation to the reference standard for anti-HPV16 E6 concentration. B–dilutional linearity for seven samples in relation to the reference standard for anti-HPV16 E7 concentration. C–dilutional linearity for seven samples in relation to the reference standard for anti-HPV18 E6 concentration. D–dilutional linearity for seven samples in relation to the reference standard for anti-HPV18 E7 concentration. E–Dilutional linearity of the reference serum over three assays runs plotted as ECL signal (left axis; solid lines) or percent recovery (right axis; dashed lines) ± %CV as a function of dilution factor of serum.

Article Snippet: Type-specific mouse monoclonal antibodies to HPV16/18 E6 (Clone C1P5, Santa Cruz Biotechnology, Santa Cruz, USA), HPV16 E7 (Clone 716–281, ThermoFisher Scientific, South San Francisco, USA), HPV18 E6 (Clone G-7, Santa Cruz Biotechnology, Santa Cruz, USA), and HPV18 E7 (Clone F-7, Santa Cruz Biotechnology, Santa Cruz, USA) were purchased for specificity experiments.

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18

doi: 10.1371/journal.pone.0229672

Figure Lengend Snippet: Estimated assay upper limit of quantitation (ULOQ) and percent recovery for type-specific anti-HPV concentrations from reference serum.

Article Snippet: Type-specific mouse monoclonal antibodies to HPV16/18 E6 (Clone C1P5, Santa Cruz Biotechnology, Santa Cruz, USA), HPV16 E7 (Clone 716–281, ThermoFisher Scientific, South San Francisco, USA), HPV18 E6 (Clone G-7, Santa Cruz Biotechnology, Santa Cruz, USA), and HPV18 E7 (Clone F-7, Santa Cruz Biotechnology, Santa Cruz, USA) were purchased for specificity experiments.

Techniques: Quantitation Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18

doi: 10.1371/journal.pone.0229672

Figure Lengend Snippet: Estimated assay lower limit of quantitation and percent recovery for type-specific anti-HPV concentrations of the reference serum.

Article Snippet: Type-specific mouse monoclonal antibodies to HPV16/18 E6 (Clone C1P5, Santa Cruz Biotechnology, Santa Cruz, USA), HPV16 E7 (Clone 716–281, ThermoFisher Scientific, South San Francisco, USA), HPV18 E6 (Clone G-7, Santa Cruz Biotechnology, Santa Cruz, USA), and HPV18 E7 (Clone F-7, Santa Cruz Biotechnology, Santa Cruz, USA) were purchased for specificity experiments.

Techniques: Quantitation Assay, Concentration Assay

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A ) Expression of gp91 phox in human induced pluripotent stem cells (iPS)-derived macrophage deficient for EROS (lanes are triplicate). ( B ) Diagram depicting the different stages of gp91 phox biosynthesis and formation of the heterodimer with p22 phox . ( C ) gp91 phox expression upon co-expression of gp91 phox -GFP and EROS-FLAG vectors compared to gp91 phox GFP alone in non-adherent HEK293-F. ( D ) Abundance of the 58 kDa form of gp91 phox in mouse NIH3T3 cells upon co-expression of gp91 phox and EROS human constructs. ( E ) Expression of p22 phox in HEK293 cells co-transfected with p22 phox and EROS mouse constructs compared to p22 phox vector alone (lanes are triplicate). ( F ) Expression level of EROS in bone marrow-derived macrophages (BMDM) from gp91 phox knockout (KO) mice compared to control mice. Data are representative of three independent experiments. ( G ) Histogram derived from gp91 phox stability curve in absence (blue) or presence (red) of EROS (four independent experiments; p-value was determined using unpaired Student’s t -test; error bars indicate SD). See also . Figure 1—figure supplement 1—source data 1. Raw unedited blots for . Figure 1—figure supplement 1—source data 2. Raw unedited blots for . Figure 1—figure supplement 1—source data 3. Uncropped gels used for .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Expressing, Derivative Assay, Construct, Transfection, Plasmid Preparation, Knock-Out, Control

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A–C ) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Construct, Transfection, Expressing, Western Blot, Quantitation Assay, Plasmid Preparation, Control, Transduction, Knock-Out

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A ) Interaction of endogenous gp91 phox and EROS analysed by immunoprecipitation (IP) of gp91 phox followed by immunoblot of EROS in PLB985 cells. Immunoblot with p22 phox serves as positive control (dashed lines indicate different exposure time). ( B ) Luminescence production from the eight different combinations of gp91 phox ( CYBB ) and EROS ( CYBC1 ) tagged with the large (L: LgBiT) fragment or small (S: SmBiT) fragment of the luciferase compared to combination with the Halo-Tag SmBiT (HT-S) control vector in HEK293 cells. Red box indicates selected constructs pair. ( C ) Nanoluc Binary Technology (NanoBiT) assay in HEK293 cells using gp91 phox ( CYBB ) and EROS ( CYBC1 ) encoded in a single construct (L- CYBC1-CYBB -S; see ‘Methods’); L- CYBC1 co-transfected with HT-S is the negative control. ( D ) HEK293 transfected with the indicated constructs were treated (right panel) or not (left panel) with succinyl acetone prior to analysis of NOX2 (gp91 phox ) expression. ( E–G ) Effect of EROS on the abundance of NOX2 homologues NOX1 ( E ), NOX4 ( F ), and NOX5 ( G ) in HEK293 or HEK293-F transfected with the indicated tagged constructs and treated or not with succinyl acetone (10 µg/mL). ( H ) Yeast 2 Hybrid analysis of EROS direct binding to NOX proteins family. ( I ) Expression of nox1 and nox4 in the specified tissues taken from control and EROS knockout (KO) mice (n = 4 biological replicates). Data are representative of three independent experiments; error bars indicate SEM of triplicates. See also . Figure 2—figure supplement 1—source data 1. Raw unedited blots for . Figure 2—figure supplement 1—source data 2. Raw unedited blots for . Figure 2—figure supplement 1—source data 3. Raw unedited blots for . Figure 2—figure supplement 1—source data 4. Uncropped gels used for . Figure 2—figure supplement 1—source data 5. Uncropped gels used for .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Immunoprecipitation, Western Blot, Positive Control, Luciferase, Control, Plasmid Preparation, Construct, Transfection, Negative Control, Expressing, Binding Assay, Knock-Out

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A–D ) Immunoprecipitation (IP) and size-exclusion chromatography (SEC) analysis of protein complexes associated with EROS. ( A ) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP, and p22 phox with Western blot for gp91 phox . Lysates treated with peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) served as reference; FG: fully glycosylated; PG: partially glycosylated; NG: non-glycosylated; Tot: total lysate; RT: run through; Elu: eluate. ( B ) SEC profile of EROS-IP eluate indicating protein (280 nm) and heme (414 nm) content. ( C ) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG, and endogenous p22 phox in SEC fractions 9–14 and 15–18. ( D ) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox, and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10 µg/ml). ( E ) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. ( F ) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see ‘Methods’). Halo Tag (HT)-SmBIT is the negative control; RLU: relative luminescence unit. ( G ) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. L: leucine; W: tryptophan; H: histidine; DBD: DNA binding domain of Gal4; AD: activation domain of Gal4 (see ‘Methods’). ( H ) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised, and labelled with anti-EROS and anti-calnexin antibodies. Scale bar = 5 μm. Data are representative of at least three independent experiments; error bars indicate SEM of triplicates. See also and . Figure 2—source data 1. Raw unedited blots for . Figure 2—source data 2. Uncropped gels used for .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: Diagram depicting the role of EROS in gp91 phox biosynthesis and formation of the heterodimer with p22 phox .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques:

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A ) EROS-FLAG affinity purification-mass spectrometry (AP-MS). Graph showing abundance (average number of peptide spectrum matches across four biological replicates) of all proteins identified in the FLAG AP-MS experiments (blue and red dots) versus their interactor specificity (SAINT probability score: SP). The red line marks the SP score cut-off (0.9) for high-confidence interacting proteins. Proteins (dots) above this cut-off (59) are deemed high-confidence interactors. The bait (EROS) and interacting proteins relevant to this study are shown in red. ( B ) Protein interaction network of the 59 high-confidence EROS-interacting proteins (SP >0.9). The protein interactions were derived from STRING. Coloured nodes represent proteins annotated with enriched Gene Ontology (GO) terms relevant to this study. ( C, D ) EROS and gp91 phox localization, following fixation and labelling with anti-EROS antibody, in HEK293 cells expressing STT3A-GFP and EROS constructs ( C ) or STT3A-GFP, gp91 phox -mRFP and EROS untagged constructs ( D ); scale bars = 5 μm. Graphs represent the intensity profile of STT3A-GFP and EROS signal or STT3A-GFP, gp91 phox -mRFP, and EROS signal measured across the nuclear membrane (indicated in red line). ( E ) Expression of gp91 phox in HEK293 cells transfected with the indicated constructs and treated with OST inhibitor (NGI-1) at the indicated concentration. ( F ) Expression of gp91 phox in control and STT3A -/- or STT3B -/- HEK293 cells transfected with the indicated vectors. ( G ) Expression of gp91 phox in PLB985 cell line treated with NGI-1 at the indicated concentration. Data are representative of three independent experiments. See also and . Figure 3—source data 1. Table of the 59 proteins identified in EROS IP-MS interactome. Figure 3—source data 2. Raw unedited blots for . Figure 3—source data 3. Uncropped gels used for .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Derivative Assay, Expressing, Construct, Membrane, Transfection, Concentration Assay, Control

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A ) gp91 phox expression in control, STT3A -/- , STT3B -/- HEK293 transfected with the indicated constructs and treated with 10 µM NGI-1 or 2 µg/mL tunicamycin. ( B ) Expression level of the ER-stress marker BiP in control and STT3A -/- or STT3B -/- HEK293 cells transfected with the indicated constructs. ( C ) Immunoblot control of STT3A expression in control and STT3A -/- or STT3B -/- HEK293 cells; Exp: experiment. ( D ) Expression level of BiP upon treatment of PLB985 cells with different concentrations of the glycosylation inhibitors NGI-1 or tunicamycin. Data are representative of at least two independent experiments. See also . Figure 3—figure supplement 1—source data 1. Raw unedited blots for . Figure 3—figure supplement 1—source data 2. Uncropped blots for .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Expressing, Control, Transfection, Construct, Marker, Western Blot, Glycoproteomics

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet: ( A–D ) P2X7 expression analysed by Western blotting of macrophages isolated from control, EROS knockout (KO) ( A ) and gp91 phox KO mice ( B ), induced pluripotent stem cells (iPS)-derived macrophages control or EROS-deficient ( C ) and of control PLB985 cells and an EROS-deficient clone ( D ). ( E, F ) P2X7 expression in RAW264.7 cells overexpressing a FLAG-tagged EROS vector ( E ) and in HEK293 cells transiently expressing the specified constructs ( F ). ( G, H ) Interaction between EROS and P2X7 probed by immunoprecipitation (IP) of EROS from RAW264.7 EROS-FLAG macrophages followed by immunoblot (IB) for P2X7 ( G ) and by Nanoluc Binary Technology (NanoBIT) assay in live HEK293 cells expressing the LgBIT-fused EROS vector with a SmBIT-fused P2X7 vector ( H ). ( I ) P2X1 expression in macrophages isolated from EROS KO mice compared to control. n = 5 biological replicates. ( J ) P2X1 abundance upon co-transfection with EROS construct in HEK293 cells. Data are representative of hree independent experiments; error bars indicate SEM of triplicates. See also and . Figure 5—source data 1. Raw unedited blots for . Figure 5—source data 2. Raw unedited blots for . Figure 5—source data 3. Raw unedited blots for . Figure 5—source data 4. Uncropped gels used for .

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Expressing, Western Blot, Isolation, Control, Knock-Out, Derivative Assay, Plasmid Preparation, Construct, Immunoprecipitation, Cotransfection

Journal: eLife

Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

doi: 10.7554/eLife.76387

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-gp91 phox (mouse monoclonal) Anti-p22 phox (rabbit polyclonal) Anti-p22 phox (mouse monoclonal) , Santa Cruz Biotechnology , sc-130543 sc-20781 sc-130550 , (1:2000) (1:1000) (1:500).

Techniques: Control, Knock-Out, Generated, Over Expression

Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: PARP1, Ku80, and Ku70 are dramatically elevated in patients with aggressive HBL and bind to the core sequence of ALCDs. a The core 18BPS was linked to biotin beads and was incubated with nuclear extracts isolated from HBL with low and high levels of TSPs. Coomassie staining identified four proteins (shown on the right) that specifically interacted with the 18BPS in nuclear extracts from aggressive HBL. b Large-scale isolation and mass spec analysis of the interacting proteins. The proteins which are specifically interacting with 18BPS are PARP1, Ku80, Ku70, and several additional proteins, some of them belong to nuclear matrix (Supplementary Fig. ). c , d Expression of PARP1, Ku80, and Ku70 is dramatically increased in patients with aggressive HBL. QRT-PCR ( c ) and western blotting ( d ) were performed with mRNAs and proteins isolated from HBL samples with low and high TSPs levels. SHP is a small heterodimer partner, which was detected by re-probe of the PARP1 membrane and which serves as a good control for protein loading. e PARP1, Ku80, and Ku70 form a complex in livers of patients with aggressive HBL. Ku70 and Ku80 were precipitated and PARP1 and Ku70 or Ku80 were determined in these IPs. IgG: heavy chains of IgG. f Fractionation of nuclear proteins from background (blue) and HBL (black) sections of aggressive HBLs by HPLC-based size exclusion chromatography (SEC). Red arrow shows elevation of optical density in the area of high MW protein–protein complexes. g Top: Examination of PARP1/Ku80/Ku70 complexes in SEC fractions. Western blotting shows the amount of proteins in each fraction. Bottom: PARP1-IP shows immunoprecipitation of PARP1 and analysis of Ku70 in these IPs. Images below show hypothetical compositions of PARP1 complexes. Error bars represent standard error of the mean ( c )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Sequencing, Incubation, Isolation, Staining, Mass Spectrometry, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Control, Fractionation, Size-exclusion Chromatography, Immunoprecipitation

Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: ALCDs are activated in aggressive HBL samples and in hepatoblastoma cancer cells by PARP1/Ku80/Ku70 complexes. a ChIP analysis of the ten representative ALCDs and two negative controls, which contain the inactive 250 bp domain (shown on the left and right) in background and HBL sections of the livers. M marker, In input, B beads, PP1 PARP1, H3K9-Ac histone H3 acetylated at K9, 3-me histone H3 trimethylated at K9. b Quantitative presentation of ChIP analysis. Amounts of PARP1/Ku80/Ku70 complexes were calculated as average of PARP1, Ku80, and Ku70 signals and then as percentage of this average signal to input. c ChIP analysis of ten representative ALCDs in HepG2 cells with and without treatment with 100 µM DPQ. d Quantitative presentations of ChIP analysis. Calculations were performed as described above. e Cell proliferation assay in HepG2, Huh6, and B6-2 cells treated with 100 µM DPQ for 72 h. Representative images highlighting changes in cell proliferation after DPQ treatment are shown in Supplementary Fig. . f Inhibition of PARP1 by DPQ reduces PARP1/Ku80/Ku70 complexes in HeLa and in HepG2 cells and reduces expression of TSPs and cell cycle proteins. Cells were treated with DPQ for 24 and 48 h and western blotting and Co-IPs were performed as described above. Error bars represent standard error of the mean ( e )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Marker, Proliferation Assay, Inhibition, Expressing, Western Blot

Journal: Communications Biology

Article Title: PARP1 activation increases expression of modified tumor suppressors and pathways underlying development of aggressive hepatoblastoma

doi: 10.1038/s42003-018-0077-8

Figure Lengend Snippet: Inhibition of PARP1 by FDA-approved inhibitor DPQ and by si-PARP1 siRNA eliminates PARP1 complexes leading to silence of ALCDs and to inhibition of cell proliferation. a Western blotting of proteins isolated from Huh6 cells treated with 100 µM DPQ for 48 h. Ku80-IP: Ku80 was immunoprecipitated and PARP1 was examined in these IPs. Bar graphs below show levels of proteins as ratios to β-actin. b HepG2 and Huh6 cells were transfected with siRNA-targeting PARP1. Expression of proteins shown on the left and right was analyzed by western blotting. Ku80-IP: western shows the disruption of PARP1/Ku80/Ku70 complexes by inhibition of PARP1. c Cell proliferation assay of HepG2, Huh6, and B6-2 cells transfected with si-PARP1. The assay was performed 48 h after transfection of siRNA. d Representative images highlighting changes in cell proliferation with si-PARP1 transfection in B6-2 cells. e Scratch assay of proliferation of HepG2 cells untreated (con) and treated with DPQ. Percentage shows % of not-closed scratches (inhibition of proliferation) at 48 h after scratch. f Cell proliferation assay shows inhibition of proliferation of HepG2 cells by low concentrations of DPQ and olaparib (Ola). Right image shows Co-IP of PARP1/Ku80/70 complexes and western blotting of downstream targets of ALCDs. g ChIP assay of the ALCD regions of within C/EBPα, HACE1, p53, and β-catenin genes in control and olaparib-treated HepG2 cells. Experiment was performed as described in legend to Fig. . Bottom images show quantitation of ChIP for ALCDs in these genes. h A diagram showing hypothesis which is based on the results of these studies. Aggressive liver cancer is associated with elevation of PARP1, which forms complexes with Ku80 and Ku70 and subsequent chromatin remodeling around ALCDs that leads to a dramatic activation of multiple pathways of liver cancer. An important part of this hypothesis is that certain TSPs are also activated by PARP1-ALCDs axis, but they are posttranslationally modified and are converted into proteins with potential oncogenic activities. Oncogenic activities of posttranslationally modified C/EBPα are shown in our recent publication . Error bars represent standard error of the mean ( c , f )

Article Snippet: Human: β-actin: Hs01060665_g1, NR1H4: Hs01026590_m1, PSMD10: Hs01100439_g1, CYP3A4: Hs00604506_m1, PCK1: Hs01572978_g1, POU5F1: Hs04260367_gH, EPCAM: Hs00901885_m1, THY-1: Hs00264235_s1, AFP: Hs00173490_m1, HNF4α: Hs00230853_m1, RB1: Hs01078066_m1, TP53: Hs01034249_m1, CELF1: Hs00198069_m1, CEBPα: Hs00269972_s1, ALB: Hs00910225_m1, CDKN1A: Hs00355782_m1, RUNDC1: Hs00405433_m1, HACE1: Hs00410879_m1, MYO18B: Hs00261714_m1, PGAP1: Hs01088726_m1, REG3A: Hs01055563_gH, PARP1; Hs00242302_m1, Ku70: Hs01922655_g1, and Ku80: Hs00897854_m1.

Techniques: Inhibition, Western Blot, Isolation, Immunoprecipitation, Transfection, Expressing, Disruption, Proliferation Assay, Wound Healing Assay, Co-Immunoprecipitation Assay, Control, Quantitation Assay, Activation Assay, Modification

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exosomal mRNAs for Angiogenic-Osteogenic Coupled Bone Repair.

doi: 10.1002/advs.202302622

Figure Lengend Snippet: Figure 2. Characterization of EVs and enriched RNAs in small EVs (sEVs). A) Surface markers (CD9, CD63, TSG101, and ARF6) and particle size distributions of EV subpopulations after purification and isolation by tangential flow filtration (TFF) and size exclusion chromatography (SEC). B) RNA distribution in different fractions of EVs. C) Particle size distributions and surface markers of isolated blank sEVs (b-sEVs), engineered sEVs (sEVsPBS), and therapeutic sEVs (t-sEVsBone RNAs) generated from blank hAdMSCs and hAdMSCs transfected by PBS buffer or bone-related pDNA cocktail, respectively. D) SEM, transmission electron microscopy (TEM), and cryogenic electron microscopy (cryo-EM) images of b-sEVs and t-sEVsBone RNAs with enriched RNAs. SEM and TEM images of b-sEVs and t-sEVsBone RNAs show no difference in the morphology of sEVs, while cryo-EM analysis suggests t-sEVsBone RNAs contain a higher RNA content. E) RNA quantity and distribution in 1 × 1012 b-sEVs, e-sEVsPBS, and t-sEVsBone RNAs (EV fraction 9–15). Synthetic mRNAs of BMP-2 and VEGF-A were used as standard samples (100 ng each mRNA). F) RT–qPCR analysis of BMP-2 and VEGF-A mRNAs indicates that t- sEVsBone RNAs contain more transcribed BMP-2 and VEGF-A mRNAs than e-sEVssPBS and b-sEVs. *p < 0.05 and ****p < 0.0001. G) Schematics of a single- sEV biochip for exosomal mRNA detection and representative total internal reflection fluorescence microscope (TIRFM) images for t-sEVsBone RNAs. Red dots: sEVs with VEGF-A mRNAs; green dots: sEVs with BMP-2 mRNAs; yellow dots: sEVs with both mRNAs (Scale bar: 10 μm). H) Colocalization percentage of t-sEVsBone RNAs with both VEGF-A and BMP-2 mRNAs (n = 15). *p < 0.05 and ****p < 0.0001. All data are presented as mean ± SD. Student’s t-test was used for comparison.

Article Snippet: After washing with PBS, the surface was incubated with a biotinylated capture antibody cocktail (20 μg mL−1 human CD63 Antibody (R&D Systems) and 20 μg mL−1 human CD9 Antibody (R&D Systems) (Table S8, Supporting Information) for 1 h at RT on the rocker.

Techniques: Isolation, Size-exclusion Chromatography, Generated, Transfection, Transmission Assay, Electron Microscopy, Cryo-EM Sample Prep, Quantitative RT-PCR, Microscopy, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exosomal mRNAs for Angiogenic-Osteogenic Coupled Bone Repair.

doi: 10.1002/advs.202302622

Figure Lengend Snippet: Figure 3. Enhanced therapeutic sEV (t-sEV) release regulated by mTORC1-autophagy axis. A) Cell-electron microscopy (Cell-EM) sections of TM-nanoEP stimulated hAdMSCs with or without plasmids show the changes of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) versus untreated hAdMSCs as a control. Quantification of cellular cryo-EM sections suggests TM-nanoEP induced activity of EV precursors, B,C) MVBs and ILVs, as evidenced by a twofold increase of the MVB number and an eightfold increase of the ILV number (n = 5). D) mRNA cargoes (BMP-2 and VEGF-A) were recognized by MB-FISH (molecular beacon-based fluorescence in-situ hybridization) probes (green). The late endosome/MVBs in hAdMSCs were stained by florescence-labeled anti-Rab7 (red). E) Colocalization quantification of mRNAs in late endosome/MVBs (n = 10). F) Western blot shows TM- nanoEP suppresses mTORC1 activity (phospho-S6 (pS6)) and thus activates autophagic activity (light chain 3-II (LC3-II). Increasing TM-nanoEP voltages results in greater reduction of mTORC1 activity and enhanced autophagy. G) Illustration of mTORC1-autophagy axis in regulating enhanced t-sEV release by TM-nanoEP. H) Western blot shows suppressed PS6 and enhanced LC3-II expression with significantly increased intracellular expression sEV marker proteins (CD63 and CD9) in wide-type hAdMSCs after TM-nanoEP. The mTORC1 and autophagic activities in TSC1/2−/−hAdMSCs were insensitive to TM-nanoEP with no impact on sEV release. I,J) sEV number and exosomal mRNA expression by TM-nanoEP stimulated TSC1/2+/+ and TSC1/2−/−

Article Snippet: After washing with PBS, the surface was incubated with a biotinylated capture antibody cocktail (20 μg mL−1 human CD63 Antibody (R&D Systems) and 20 μg mL−1 human CD9 Antibody (R&D Systems) (Table S8, Supporting Information) for 1 h at RT on the rocker.

Techniques: Electron Microscopy, Control, Cryo-EM Sample Prep, Activity Assay, In Situ Hybridization, Staining, Labeling, Western Blot, Expressing, Marker

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 initially delays Delta16HER2-driven tumorigenesis, but then promotes a high tumor multiplicity. ( A ) Breeding scheme used to obtain Delta16HER2/SIRT6-OE female mice and a representative genotyping PCR. ( B ) Kaplan-Meir curves comparing the percentage of tumor-free mice between Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 17/group). ( C ) Representative post mortem pictures of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age (top panels), and relative H&E staining of the primary mammary tumor masses (bottom panels). Black arrows indicate tumor foci. ( D , E ) Tumor multiplicity and tumor growth curves of Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 17/group). ( F ) mRNA expression of Sirtuin1-7(Sirt1-7) and Delta16HER2 normalized to β-Actin mRNA level in tumors of 30-week-old Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 4/group). ( G ) Representative images of IHC staining for SIRT6 (brown) in tumors of 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice, respectively. Delta16HER2/SIRT6-OE tumors show a strong SIRT6 level and mainly localized into the nucleus. Scale bar, 25 mm. ( H , I ) Western blot analysis and relative quantification of total SIRT6 and phosphorylated SIRT6 (pSIRT6-Ser388) normalized to β-Actin protein level. pSIRT6-Ser388/total SIRT6 represents the ratio of β-Actin-normalized phosphorylated protein over the total SIRT6 protein. ( J ) Flow cytometry staining for Delta16HER2 and SIRT6 of primary cells (passage 4 in vitro) derived from tumors of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age. MFI, Median Fluorescence Intensity. The experiment was carried out in triplicates. In ( B ) ***p = 0.0008 (Log-rank test); in ( D , E ) **p < 0.01, ***p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparisons test); in ( F , H , J ) *p < 0.05, **p < 0.01 (two-tailed unpaired t test). Error bars represent SD. See also Fig. .

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Quantitative Proteomics, Flow Cytometry, In Vitro, Derivative Assay, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 boosts tumor cell migration, invasion and lung metastasis in Delta16HER2/SIRT6-OE mice. ( A ) Representative pictures of H&E staining on lungs of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age (n = 7/group). Dashed black lines highlight the metastatic area in each picture. Quantification is represented as number of metastasis per mouse ( B ) and as metastasis area over total section area ( C ). Both number and area of metastasis were calculated as average over 2–6 consecutive tissue sections per mouse. ( D ) Soft agar assay performed on primary cells (passage 4 in vitro) derived from tumors of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age. Stereomicroscope images, 4X magnification (top) and 10X zoom (bottom). Number of colonies ( E ) were quantified using ImageJ software. Results are expressed as % of total number of seeded cells. The assay was performed in triplicates. ( F , G ) Transwell migration assay and quantification of Delta16HER2 and Delta16HER2/SIRT6-OE primary cells. Pore size 8.0 mm. Quantification is reported as percentage of migrating cells over the total number of seeded cells. In ( B , C , E , G ) *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t test). Error bars represent SD.

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: Migration, Staining, Soft Agar Assay, In Vitro, Derivative Assay, Software, Transwell Migration Assay, Pore Size, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 prevents tumor cells from Delta16HER2-induced G2/M arrest and senescence. ( A ) Ex vivo cell cycle analysis of cell suspensions derived from Delta16HER2 and Delta16HER2/SIRT6-OE tumors at 20 weeks of age (top panels) and at 30 weeks of age (bottom panels). DNA content was evaluated by flow cytometry using propidium iodide (PI) staining (n = 3) and then analyzed using Dean-Jett-Fox algorithm in FlowJo software. Percentages of cells in each cell cycle phase are summarized in each panel. ( B ) Immunoblot image and quantification of Cyclin D1 and Cyclin E levels normalized to β-Actin in tumors of either 20- (top) or 30-week-old (bottom) Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4). ( C ) mRNA expression of Trp53 , Cdkn2a and Cdkn1a genes normalized to β-Actin mRNA level in tumors of 30-week-old Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 4/group). ( D ) Detection of senescence-associated β-galactosidase (SA-β-Gal) activity in frozen tumors of 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE (n = 3). Quantification is expressed as % of SA-β-Gal positive area (blue) with respect to the total section area. Scale bar, 25 mm. In ( B – D ) ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t test). Error bars represent SD. See also Fig. .

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: Ex Vivo, Cell Cycle Assay, Derivative Assay, Flow Cytometry, Staining, Software, Western Blot, Expressing, Activity Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 promotes stemness and self-renewal capacity of Delta16HER2 tumor cells. ( A – D ) Flow Cytometry analysis of CD44, CD24, OCT3/4, Notch1 and NANOG levels in primary tumor cells derived from 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4). Quantification is expressed as median fluorescence intensity (MFI) ( A ) and as percentage of positive cells ( C and D ). ( E ) Representative images of mammosphere formation assay performed on primary tumor cells derived from 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4, top panel). Quantification is represented for 2 serial cloning assays and is expressed as percentage of mammosphere forming units (MFU) with respect to the total number of seeded cells (bottom panel). See also Fig. . ( F ) Flow cytometry analysis of SIRT6 and HER2 expression in CAM6 cells stably transduced with pLENTI-Blank (empty vector) and pLENTI-SIRT6. ( G ) Mammosphere formation assay on CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6. Scale bar, 200 mm. ( H , I ) Quantification of mammosphere forming capacity (MFU %) and average sphere area of CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6 cells. Experiment was carried out in triplicate. ( J ) Immunofluorescence staining of OCT3/4 on CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6 mammospheres. DAPI is used for nuclei counterstaining. Magnification 40X. In ( A , C – E , H , I ) *p < 0.05, **p < 0.01, ***P < 0.001 (two-tailed unpaired t test). Error bars represent SEM.

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: Flow Cytometry, Derivative Assay, Fluorescence, Tube Formation Assay, Cloning, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Immunofluorescence, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 is amplified in a subset of HER2-positive breast cancer patients and correlates with poor relapse-free-survival. ( A ) cBioPortal data summarizing the type and frequency of alteration of HER2 ( ERBB2 ) and SIRT6 genes in sequenced samples from publicly available invasive breast cancer studies (listed in the “study of origin” section). A total of 4860 and 4379 breast cancer patients were profiled for ERBB2 and SIRT6 alterations, respectively. ( B ) Co-occurrence/Mutual exclusivity analysis from cBioPortal indicating the number of patient samples that have amplification of HER2 (ERBB2) and SIRT6 alone, in both or neither genes. Odd ratio = neither*both/SIRT6 not ERBB2*ERBB2 not SIRT6. Log2 Odd Ratio > 1 indicates co-occurrence of HER2 and SIRT6 amplifications (p < 0.0001, Pearson correlation). ( C – E ) Kaplan–Meier plots from GOBO database using overall survival ( C ) and relapse-free survival (RFS) ( D , E ) as outcome in all HER2-enriched breast cancer ( D ) and in grade 3 HER2-enriched breast cancer ( E ). Data have been stratified into the two quantiles based on SIRT6 gene expression level (SIRT6_low, grey line and SIRT6_high, red line) using 10-year censoring as endpoint. ( F ) DNA microarray data from bc-GenExMiner showing SIRT6 expression in HER2- (n = 4068) and HER2 + (n = 680) breast cancer samples. ( G , H ) Kaplan–Meier plots from bc-GenExMiner using distant metastasis free survival (DMFS) and disease free survival (DFS) as outcomes in HER2-enriched breast cancer. Data have been stratified into the two quantiles based on SIRT6 gene expression level (SIRT6_low, purple line and SURT6_high, blue line). HR (hazard ratio) and 95% CI (confidence interval) are reported in the figure. In ( C , D , F , G ) *p = 0.02436, *p = 0.02143, *p = 0.0468, *p = 0.0222 (Log-rank test). In ( E ) *p = 0.0322 (Welch’s test). See also Figs. and .

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: Amplification, Gene Expression, Microarray, Expressing

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: ChIP- and RNA-seq identify TBX3 as a SIRT6 target and a prognostic marker in HER2 + breast cancer. ( A ) Venn diagram representing the genes that are differentially expressed and differentially bound by H3K9ac (cutoff FDR ≤ 0.05) in tumors from Delta16HER2/SIRT6-OE versus Delta16HER2 controls (20 weeks of age, n = 2 for RNA-seq and n = 2–3 for ChIP-seq). ( B , C ) ChIP and RNA-seq tracks showing H3K9ac level and expression for Il12a and Tbx3 genes in Delta16HER2 (blue) versus Delta16HER2/SIRT6-OE (red) tumors (at 20 weeks of age). ( D ) Bee swarm plots computed in bc-GenExMiner showing the expression levels of IL12A and TBX3 in HER2–/ + breast cancers. ( E , F ) Kaplan–Meier plots from bc-GenExMiner using distant metastasis free survival (DMFS) and disease free survival (DFS) as outcomes in HER2-enriched breast cancer. Data have been stratified into the two quantiles based on IL12A gene expression level (( E ), IL12A_low, purple line and IL12A_high, blue line) and TBX3 gene expression level (( F ), TBX3_low, purple line and TBX3_high, blue line). HR (hazard ratio) and 95% CI (confidence interval) are reported in the figure. In ( D ) ****P < 0.0001 (Welch’s test). In ( E , F ) ns, p = 0.1124; ns, p = 0.7269; ***p = 0.0004, **p = 0.0075 (Log-rank test). See also Figs. and .

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: RNA Sequencing, Marker, ChIP-sequencing, Expressing, Gene Expression

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6-OE induces loss of TBX3 and aggressiveness in both mouse and human models of Delta16HER2/HER2 + . ( A ) Representative pictures and ( B ) quantification for Tbx3 IHC in the indicated mouse tumors. ( C ) Immunoblot of Tbx3 in mouse tumors of the indicated genotype (20 weeks, n = 4 per group). ( D ) Representative pictures of the human BT474 cell line transfected with either pHIV-dTomato or pHIV-SIRT6-dTomato. ( E ) Real-time PCR for SIRT6 and TBX3 expression in sorted BT474 transfected as indicated. Not transfected HEK293 were used as control sample and TBP as housekeeping gene. ( F ) Representative pictures and ( G ) quantification of the Transwell migration assay of the human BT474 cell line transfected with either pHIV-dTomato or pHIV-SIRT6-dTomato. ( H ) Representative pictures and ( I ) number and area quantification of pHIV-dTomato and pHIV-SIRT6-dTomato BT474 mammospheres. MFU% = % of mammary forming units over total number of seeded cells. A.U . arbitrary unit. Bars indicate mean ± SD. In ( B ) ***p = 0.0009; ( C ) **p = 0.0077; ( D ) **p = 0.0014, ***p = 0.0003; ( G ) **p = 0.0077 and ( I ) statistics indicates unpaired two-tailed t test.

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, Control, Transwell Migration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: Loss of TBX3 mimics SIRT6 -OE in vitro and predicts poor survival of HER2-positive breast cancer patients. ( A ) Immunoblot and quantification of TBX3 and GAPDH in lysates of BT474 cell line transfected with the indicated siRNA (48 h post transfection, n = 2 independent replicates). ( B ) Representative pictures and ( C ) quantification of the Transwell migration assay of the human BT474 cell line transfected as indicated (n = 5). ( D ) Representative pictures and ( E ) number and area quantification of siRNA control and siRNA TBX3 BT474 mammospheres (n = 3). MFU% = % of mammary forming units over total number of seeded cells. A.U . arbitrary unit. Bars indicate mean ± SD. See also Fig. . ( F ) Lollipop graph from cBioPortal showing mutations of the TBX3 gene in breast cancer patients (n = 338). Y axis represents the number of mutations while X axis indicates the correspondent amino acid position. OncoKB tracks in blue indicate those mutations that are likely-oncogenic and likely loss-of-function. Types of mutation are color-coded in the figure legend. ( G ) Summary of total number and frequency of TBX3 mutations in all, HER2-WT and HER2 amplified breast cancer subsets. Pie charts indicate the frequency of the different types of TBX3 mutations over total number of TBX3 mutations in each subset. ( H ) Kaplan–Meier plots from cBioPortal showing the overall survival of patients with HER2 amplified alone (n = 271), and those with concomitant SIRT6 amplification (n = 8) or TBX3 loss-of-function mutations (n = 20) . P = 0.0007 (Log-rank Mantel-Cox test). In ( A ) **p = 0.0047, ( C ) *p = 0.0282, ( E ) **p = 0.0051 and ns, p = 0.2106 indicates unpaired two-tailed t test.

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: In Vitro, Western Blot, Transfection, Transwell Migration Assay, Control, Mutagenesis, Amplification, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: qRT-PCR primer list.

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: Sequencing, Amplification

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: Summary of used antibodies.

Article Snippet: SIRT6 , Rabbit monoclonal anti-sirt6 , WB , 1:1000 , Cell signaling technology.

Techniques: