Journal: Breast Cancer Research : BCR

Article Title: The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth

doi: 10.1186/s13058-024-01778-w

Figure Lengend Snippet: NUDT5 expression in breast cancer. A NUDT5 mRNA expression levels from publicly available databases (from The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium [METABRIC]). The mRNA expression level of NUDT5 is compared between TNBC and ER-positive breast cancers, as well as TNBC and normal breast. B NUDT5 mRNA expression levels across the different PAM50 subgroups using the METABRIC data. The mRNA expression level of NUDT5 is compared between basal subtype and the normal-like, Luminal A, Luminal B, HER2-enriched, and Claudin-low subtypes. C NUDT5 expression across the different triple-negative breast cancer (TNBC) subtypes (from Burstein et al. ) using data from METABRIC. D NUDT5 mRNA expression in non-TNBC and TNBC cell lines using data from the Cancer Cell Line Encyclopedia is shown . E NUDT5 protein expression is shown by Western blot analysis of multiple cell lines, including normal-like, estrogen receptor-positive, and TNBC cell lines. Quantification of NUDT5 protein levels in non-TNBC and TNBC cells is also shown. The differences of NUDT5 expression levels between two subtypes were determined by Student’s t test and the differences among multiple subtypes were determined by one-way ANOVA. ( ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001)

Article Snippet: NUDT5-specific targeting siRNA oligos were purchased from Sigma-Aldrich (SASI_Hs01_00109215, SASI_Hs02_00345134, 3’UTR siRNA: 5′ UGA AAG GGC UCU CCA GAU A 3′; St. Louis, MO).

Techniques: Expressing, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth

doi: 10.1186/s13058-024-01778-w

Figure Lengend Snippet: Loss of NUDT5 induces oxidative 8-oxoG and DNA damage response. A 8-oxoG lesions were stained in TNBC (MDA-MB-231) and ER-positive (MCF-7) cells treated with siLuc or siNUDT5, and nuclei were counterstained with DAPI after 4 days. The data is shown as nuclear intensity for siLuc- or siNUDT5-treated cells. B γH AX was stained in MDA-MB-231 and MCF-7 cells treated with siLuc or siNUDT5, and nuclei were counterstained with DAPI after 7 days. The data is shown as γH AX positivity, and was compared between the different treatments. Statistical significance was analyzed by the Student’s t test. C Representative DNA fibers from MDA-MB-231 and MCF-7 cells treated with siLuc or siNUDT5. Cells were first labelled with 30 min treatment with 50 µM IdU, followed by 30 min treatment with 100 µM CIdU. DNA fiber length was imaged by an Andor Revolution XDi WD Spinning disk confocal microscope and analyzed with Imaris software. The fork speed = (the length of IdU labeled DNA fiber + the length of CdU labeled DNA fiber)/total labeling time (1 h). The difference between siLuc- and siNDUT5-treated DNA fibers is shown graphically and compared using a Student’s t test. Additional TNBC (MDA-MB-436, MDA-MB-468) and ER-positive (ZR-75-1 and MDA-MB-361) cell lines are shown in Additional file , : Figures S7 and S8. Proof of effective knockdown is shown via Western blot and qPCR in Additional file : Figure S2A

Article Snippet: NUDT5-specific targeting siRNA oligos were purchased from Sigma-Aldrich (SASI_Hs01_00109215, SASI_Hs02_00345134, 3’UTR siRNA: 5′ UGA AAG GGC UCU CCA GAU A 3′; St. Louis, MO).

Techniques: Staining, Microscopy, Software, Labeling, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth

doi: 10.1186/s13058-024-01778-w

Figure Lengend Snippet: Proposed mechanism of NUDT5 biological function in breast cancers. The level of ROS is low in ER-positive tumors, resulting in low accumulation of 8-oxoG and γH 2 AX lesions in the nucleus. In such cases, the inhibition or loss of NUDT5 does not affect the growth of these ER-positive tumors. In TNBC tumors, both ROS and NUDT5 levels are elevated. When NUDT5 is abundant, it mitigates oxidative DNA damage by hydrolyzing oxidized deoxyribonucleoside diphosphates. Consequently, there is no incorporation of 8-oxoG into the DNA, and γH 2 AX lesions do not accumulate. However, in the absence of NUDT5, uncontrolled oxidative stress on the nucleotide pool occurs. This leads to the incorporation of 8-oxoG lesions into the DNA and the accumulation of γH 2 AX lesions in the nucleus, ultimately causing DNA replication fork slowing and reduced proliferation

Article Snippet: NUDT5-specific targeting siRNA oligos were purchased from Sigma-Aldrich (SASI_Hs01_00109215, SASI_Hs02_00345134, 3’UTR siRNA: 5′ UGA AAG GGC UCU CCA GAU A 3′; St. Louis, MO).

Techniques: Inhibition

Journal: Breast Cancer Research : BCR

Article Title: The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth

doi: 10.1186/s13058-024-01778-w

Figure Lengend Snippet: NUDT5 loss inhibits triple-negative breast cancer growth. A Cell growth of estrogen receptor-positive breast cancer cells (MCF7, ZR-75-1 and MDA-MB-361), and triple-negative breast cancer cells (MDA-MB-231, MDA-MB-436, and MDA-MB-468) following siLuc or siNUDT5 treatment. Knockdown efficiency of protein samples harvested at Day 7 is shown by Western blot analysis (protein expression at D1 and D7 is also shown in Additional file : Figure S2A). RNA expression after siRNA knockdown at D1 and D7 is also shown in Additional file : Figure S2A. The significant differences between day 7 cell counts were determined using Student’s t test ( ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Shown in this figure is one representative growth experiment for each cell line. This experiment was repeated in each of these cell lines with similar results showing effective siRNA knockdown (at the RNA and protein levels), as well as effective growth suppression in the TNBC cell lines

Article Snippet: NUDT5-specific targeting siRNA oligos were purchased from Sigma-Aldrich (SASI_Hs01_00109215, SASI_Hs02_00345134, 3’UTR siRNA: 5′ UGA AAG GGC UCU CCA GAU A 3′; St. Louis, MO).

Techniques: Western Blot, Expressing, RNA Expression

Journal: Breast Cancer Research : BCR

Article Title: The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth

doi: 10.1186/s13058-024-01778-w

Figure Lengend Snippet: NUDT5 depletion suppresses TNBC cell proliferation. A BrdU incorporation in the ER-positive cell line MCF-7 and TNBC cell line MDA-MB-231 treated with siLuc versus siNUDT5, or DMSO versus TH5427. Cells were treated 48 h prior to the assay. B DRAQ7 cell death assay in the ER-positive cell line MCF-7 and TNBC cell line MDA-MB-231 treated with siLuc versus siNUDT5, or DMSO versus TH5427. Cells were treated 48 h prior to the assay. 10 µM staurosporine was used as a positive control. C Annexin V/ propidium iodide (PI) staining in the ER-positive cell line MCF-7 and TNBC cell line MDA-MB-231 treated with siLuc versus siNUDT5, or DMSO versus TH5427. Cells were treated 48 h prior to the assay. 1 µM bortezomib or 10 µM staurosporine were used as a positive control. The apoptotic cell population is composed of both early (Annexin V-positive/PI-negative) and late apoptotic (Annexin V-positive/PI-positive) cells. Statistical comparisons were analyzed by the Student’s t test, and p values are shown

Article Snippet: NUDT5-specific targeting siRNA oligos were purchased from Sigma-Aldrich (SASI_Hs01_00109215, SASI_Hs02_00345134, 3’UTR siRNA: 5′ UGA AAG GGC UCU CCA GAU A 3′; St. Louis, MO).

Techniques: BrdU Incorporation Assay, Positive Control, Staining

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB triggered GSH synthesis. (A,B) The GSE101586, GSE112214, and GSE101684 data sets were re-analyzed to identify the circRNAs that were differentially expressed between the LUAD and adjacent normal tissues; (C) P4HB expression levels in LUAD tissue samples were analyzed using TCGA data; (D) the prognosis of P4HB expression was shown by Kaplan-Meier plotter curves; (E,F) the pathway enrichment of those metabolites with a high P4HB level was predicted; (G) circP4HB expression levels in LUAD tissue samples were detected by RT-qPCR assays; (H,I) the GSH levels and GSH/GSSG ratios of circP4HB overexpression H1299 and SPCA1 cells were measured. **, P<0.01; ***, P<0.001. LUAD, lung adenocarcinoma; GSH, glutathione; GSSG, oxidized glutathione disulfide.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Over Expression

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB protected LUAD from ferroptosis through promoting GSH synthesis. (A) The effect of erastin (10 µm 24 h) on the viability of LUAD and 16HBE cells was evaluated by CCK-8. DMSO was used as a control; (B,C) ferroptosis markers, Ptgs2 and MDA, were examined in cell lines in response to erastin (10 µm, 24 h); (D) the effect of erastin on the viability of H1299 cells overexpressing circP4HB was measured by CCK-8; (E,F) GSH levels and GSH/GSSG ratios of the circP4HB-overexpressing cells were measured; (G,H) Ptgs2 mRNA and MDA levels in circP4HB-overexpressing cells were examined in response to erastin; (I,J) lipid ROS and JC-1 levels were measured following immunofluorescence staining of H1299 cells following treatment with erastin. *, P<0.05; **, P<0.01; ***, P<0.001; ns, non-significant. LUAD, lung adenocarcinoma; Ptgs2, prostaglandin-endoperoxide synthase 2; MDA, malondialdehyde; DMSO, dimethyl sulfoxide; CCK-8, cell counting kit-8; GSH, glutathione; lipid ROS, lipid reactive oxygen species.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: CCK-8 Assay, Control, Immunofluorescence, Staining, Cell Counting

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: Validation of the interactions between circP4HB, miR-1184, and SLC7A11. (A) The relative abundance of circP4HB in the nucleus and cytoplasm was investigated via nuclear mass separation assays; (B) bioinformatic predictions https://circinteractome.nia.nih.gov and circNet, identified miR-1184 as a target of circP4HB; (C) FISH assay showed circP4HB and miR-1184 probes co-localized in the cytoplasm; (D) the interaction between circP4HB and miR-1184 was determined by using dual-luciferase reporter assays; (E) RIP assays were performed on the cells using AGO2 antibodies; (F) RNA pull-down assays and qRT-PCR assays were used to confirm the binding of circP4HB to miR-1184; (G) predictions using TargetScan and FerrDb, SLC7A11 was taken forward as a target of interest; (H,I) dual-luciferase reporter assays were carried out using HEK-293 T cells co-infected with a plasmid containing the SLC7A11 3'-UTR and miR-1184 mimics; (J,K) miR-1184 and SLC7A11 levels were measured in knockdown circP4HB cells. Negative controls (NC) were also used. **, P<0.01; ***, P<0.001; ns, non-significant. SLC7A11, solute carrier family 7 member 11; AGO2, Argonaute 2; RIP, RNA immunoprecipitation; FISH, fluorescence in situ hybridization.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: Biomarker Discovery, Luciferase, Quantitative RT-PCR, Binding Assay, Infection, Plasmid Preparation, Knockdown, RNA Immunoprecipitation, Fluorescence, In Situ Hybridization

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB inhibits ferroptosis by regulating miR-1184/SLC7A11-mediated GSH synthesis. (A,B) Lipid ROS and JC-1 levels were measured using Lipid ROS and JC-1 staining; (C,D) GSH levels and the GSH/GSSG ratios were detected; (E) protein levels of SLC7A11 were tested by western blot analysis; (F) mRNA levels of downstream effectors (Gclc, Gclm, Gsr, Gss, and Nqo1) were examined by qRT-PCR. +, transfected with; −, transfected without. **, P<0.01; ***, P<0.001. SLC7A11, solute carrier family 7 member 11; GSH, glutathione; lipid ROS, lipid reactive oxygen species; GSSG, oxidized glutathione disulfide.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: Staining, Western Blot, Quantitative RT-PCR, Transfection

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB promotes LUAD growth and inhibits ferroptosis in vivo. (A-C) Tumor size and, weight, and volume of the mice were measured. (D,E) Gene expression levels of circP4HB and SLC7A11 were measured by in harvested tumor tissues by qRT-PCR. (F) SLC7A11 levels were detected by IHC assays. (G) SLC7A11 mRNA expression levels in LUAD tissues and normal samples from TCGA data. (H) Kaplein-Meier plotter showing prognostic significance of SLC7A11 gene expression in LUAD patients. The prognosis of abnormal SLC7A11 expression in LUAD patients from TCGA data. (I) The SLC7A11 mRNA levels in 50 cases (tumor tissues and para-tumor samples) were detected by qRT-PCR. (J) Protein levels of SLC7A11 in LUAD tissues and para-tumor were tested by IHC. *, P<0.05; **, P<0.01; ***, P<0.001; ns non-significant. LUAD, lung adenocarcinoma; SLC7A11, solute carrier family 7 member 11; TCGA, The Cancer Genome Atlas, IHC, immunohistochemistry.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: In Vivo, Gene Expression, Quantitative RT-PCR, Expressing, Paraffin-embedded Immunohistochemistry, Immunohistochemistry

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: Schematic representation of a proposed mechanism by which circP4HB protects lung adenocarcinoma cells from ferroptosis via SLC7A11-mediated glutathione synthesis. SLC7A11, solute carrier family 7 member 11; GSH, glutathione; GSSG, oxidized glutathione disulfide; ROS, reactive oxygen species.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: