Journal: iScience

Article Title: Mutation T9I in Envelope confers autophagy resistance to SARS-CoV-2 Omicron

doi: 10.1016/j.isci.2025.112974

Figure Lengend Snippet: E T9I has increased affinity to autophagosome-associated proteins (A) Principal component analysis of the differential interactome data , the individual replicates are separated (black: GFP controls, Green: E T9 pulldown, Purple: E T9I pulldown) (B) Volcano plot of the differential interactome analysis showing enriched proteins in E T9I pulldown versus the p value (-log P). Five highly significantly enriched proteins are highlighted in red and via labels. (C–G) Quantification of proximity ligation assays between transiently expressed SARS-CoV-2 E variants 30 h post transfection in HeLa cells and endogenous SNX12, STX12, TMEM87B, ABCG2 and TAB1, as indicated. Representative images depicted. PLA signal, red. Scale Bar, 10μm. DAPI, nuclei (blue). Lines represent the mean of N = 18–59 (individual cells) ±SEM. (H) Quantification of autophagosome levels by flow cytometry in HEK293T autophagy reporter cells (HEK293T-GL) transiently expressing StrepII-tagged SARS-CoV-2 E variants (48 h post transfection) and depleted of indicated proteins by siRNA. Bars represent the mean of N = 3 (biological replicates) ±SEM. Student’s t test with Welch’s correction. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Monoclonal rabbit anti-ABCG2 Antibody (2K8X1) (WB 1:500) , Novus Biologicals , Cat#NBP3-15559.

Techniques: Ligation, Transfection, Flow Cytometry, Expressing

Journal: Biomaterials Research

Article Title: Repairing Effect and Mechanism of the 4-Dimensionally Printed Limbal Stem Cell Strategy on Corneal Alkali Burns in Large Animals

doi: 10.34133/bmr.0262

Figure Lengend Snippet: Identification of the stemness of porcine limbal stem cells (PLSCs) and evaluation of the biocompatibility of 4D-CTH. (A) Representative images of primary culture of PLSCs at different time points. (B) Dual fluorescence staining of cytokeratin 14 (CK14) and Delta Np63 alpha (ΔNp63α) to identify primary PLSCs. (C) Dual fluorescence staining of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and ATP-binding cassette sub-family G member 2 (ABCG2) to identify primary PLSCs. (D) Migration ability of 4D-CTH-encapsulated limbal stem cells (LSCs) in the flat scratch assay. (E) Representative images of live and dead cell staining of 4D-CTH- and CTH-encapsulated LSCs. (F) Quantitative analysis of healing rate in the flat scratch assay. (G) Quantitative analysis of cell viability after 4D-CTH- and CTH-encapsulated LSCs. Scale bar, 50 μm; white scale bar, 200 μm. ns, not significant. D0, D3, D5, and D7, days 0, 3, 5, and 7; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: After sealing, the plates were fixed with 4% paraformaldehyde fixative (Sigma-Aldrich, USA) at room temperature for 20 min, followed by permeabilization with 0.2% Triton X-100 (Sigma-Aldrich, USA) for 5 min. After blocking for 30 min, the following specific primary antibodies were added and incubated overnight at 4 °C: BMI1 antibody (Cell Signaling Technology, USA), ΔNp63α antibody (Cell Signaling Technology, USA), CK14 antibody (Cell Signaling Technology, USA), and ABCG2 antibody (Proteintech, China).

Techniques: Fluorescence, Staining, Binding Assay, Migration, Wound Healing Assay

Journal: Molecular Cancer Therapeutics

Article Title: Tunicamycin Potentiates Cisplatin Anticancer Efficacy through the DPAGT1/Akt/ABCG2 Pathway in Mouse Xenograft Models of Human Hepatocellular Carcinoma

doi: 10.1158/1535-7163.mct-13-0201

Figure Lengend Snippet: Figure 1. The protein level of ABCG2 can be influenced by alternative inhibitors. MHCC-97L (A) and MHCC-97H (B) cell lysates were treated with PNGase F, Endo Hf, or a-2,3-neuraminidase at 37C according to the datasheets. The shift in the ABCG2 molecular weight was detected by Western blotting. C, wild-type ABCG2 and the ABCG2 N596Q variant were stably transfected into Huh7 cells. ABCG2 expression was analyzed using Western blotting. D, ABCG2 mRNA levels were analyzed by real-time PCR with total RNA extracted from Huh7 cells expressing wild-type ABCG2 or the N596Q variant. E, MHCC-97L and MHCC-97H were treated with glycosylation inhibitors (2.5 mg/mL tunicamycin, 10 mg/mL swainsonine, and 2.5 mg/mL 2-DG) and PI3K/Akt inhibitors (2 mmol/L Wortmannin and 10 mmol/L LY294002) for 24 hours. After treatment, total protein was extracted and immunoprobed to detect ABCG2 expression. b-Actin was used as a loading control.

Article Snippet: Vector constructs, lentivirus production, and cell transfection The ABCG2 cDNA clone (SC-117127) was purchased from OriGene.

Techniques: Molecular Weight, Western Blot, Variant Assay, Stable Transfection, Transfection, Expressing, Real-time Polymerase Chain Reaction, Glycoproteomics, Control

Journal: Molecular Cancer Therapeutics

Article Title: Tunicamycin Potentiates Cisplatin Anticancer Efficacy through the DPAGT1/Akt/ABCG2 Pathway in Mouse Xenograft Models of Human Hepatocellular Carcinoma

doi: 10.1158/1535-7163.mct-13-0201

Figure Lengend Snippet: Figure 2. The NLG inhibitor tunicamycin altered ABCG2 cellular location. A, three hepatocellular carcinoma cell lines were treated with 2.5 mg/mL tunicamycin for 24 hours. The protein levels of ABCG2, Akt, and p-Akt were examined by Western blotting. B, levels of ABCG2, Akt, and p-Akt were reduced by tunicamycin in a dose- and time- dependent manner in MHCC-97L cells. C, MHCC-97L cells seeded on BD culture slides were treated with 2.5 mg/mL tunicamycin or 10 mmol/L LY294002 for 24 hours; this process was followed by immunostaining to detect the ABCG2 protein. The expression and localization of ABCG2 were observed using confocal microscopy. Representative images of ABCG2 immunostaining are shown (600 magnification). The BXP-21 monoclonal ABCG2 antibody was used for immunodetection, and the nuclei were stained with 40,6-diamidino- 2-phenylindole (DAPI).

Article Snippet: Vector constructs, lentivirus production, and cell transfection The ABCG2 cDNA clone (SC-117127) was purchased from OriGene.

Techniques: Western Blot, Immunostaining, Expressing, Confocal Microscopy, Immunodetection, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Tunicamycin Potentiates Cisplatin Anticancer Efficacy through the DPAGT1/Akt/ABCG2 Pathway in Mouse Xenograft Models of Human Hepatocellular Carcinoma

doi: 10.1158/1535-7163.mct-13-0201

Figure Lengend Snippet: Figure 4. Tunicamycin enhanced the antitumor effect of chemotherapy drugs in vitro and in vivo. A, MHCC-97L cells were treated with tunicamycin (1.0 mg/ mL), 5-FU (100 mg/mL), or CDDP (20 mg/mL) or a combination of tunicamycin and 1 of the 3 aforementioned drugs for 48 hours. Huh7 cells (B) were treated with tunicamycin (1.0 mg/mL), 5-FU (10 mg/mL), or CDDP (3.5 mg/mL) or a combination of tunicamycin and 1 of the 3 aforementioned drugs for 48 hours. The expression levels of ABCG2, Akt pathway proteins, PCNA, and cleaved PARP were examined by Western blotting. The in vivo therapeutic effects of tunicamycin and CDDP were examined in nude mouse models using MHCC-97L (C) or Huh7 (D) cells. Corresponding tumors from the 4 groups of animals bearing xenografts after treatment were excised and weighed. , P < 0.05 (Student t test).

Article Snippet: Vector constructs, lentivirus production, and cell transfection The ABCG2 cDNA clone (SC-117127) was purchased from OriGene.

Techniques: In Vitro, In Vivo, Expressing, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Tunicamycin Potentiates Cisplatin Anticancer Efficacy through the DPAGT1/Akt/ABCG2 Pathway in Mouse Xenograft Models of Human Hepatocellular Carcinoma

doi: 10.1158/1535-7163.mct-13-0201

Figure Lengend Snippet: Figure 5. The sensitization effect of tunicamycin could be rescued by forced expression of ABCG2 or Akt-myr. A, overexpression of ABCG2 in Huh7 and SMMC-7721 cells was validated by Western blotting. B, Western blotting analysis of ABCG2, PCNA, and cleaved PARP in Huh7 cells stably transfected with empty vector (pWPXL) or ABCG2 and exposed for 48 hours to tunicamycin (1.0 mg/ mL) and CDDP (3.5 mg/mL), alone or in combination. C, overexpression of Akt-myr in Huh7 and SMMC-7721 cells was validated by Western blotting. D, Western blotting analysis of p-Akt, PCNA, and cleaved PARP in Huh7 cells stably transfected with the empty vector (pWPXL) or Akt-myr and exposed for 48 hours to tunicamycin (1.0 mg/mL) and CDDP (3.5 mg/mL), alone or in combination.

Article Snippet: Vector constructs, lentivirus production, and cell transfection The ABCG2 cDNA clone (SC-117127) was purchased from OriGene.

Techniques: Expressing, Over Expression, Western Blot, Stable Transfection, Transfection, Plasmid Preparation

Journal: Molecular Cancer Therapeutics

Article Title: Tunicamycin Potentiates Cisplatin Anticancer Efficacy through the DPAGT1/Akt/ABCG2 Pathway in Mouse Xenograft Models of Human Hepatocellular Carcinoma

doi: 10.1158/1535-7163.mct-13-0201

Figure Lengend Snippet: Figure 6. DPAGT1 knockdown enhanced the antitumor effect of CDDP in hepatocellular carcinomas. A, Western blotting assays and quantification of DPAGT1 expression in 30 paired hepatocellular carcinoma tissue samples. The representative immunoblots of DPAGT1 in 10 paired tissue samples are shown. B, protein levels of DPAGT1 in hepatocellular carcinoma cell lines and the normal liver cell line L02 were detected using Western blotting. C, DPAGT1 knockdown reduced the expression of ABCG2 and p-Akt as confirmed by Western blotting. D, knockdown of DPAGT1 in combination with CDDP (3.5 mg/ mL) reduced PCNA expression and induced the protein level of cleaved PARP compared with either single treatment.

Article Snippet: Vector constructs, lentivirus production, and cell transfection The ABCG2 cDNA clone (SC-117127) was purchased from OriGene.

Techniques: Knockdown, Western Blot, Expressing

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 6. Network-based analysis of interaction network corresponding to candidate genes. (A) The node degree distribution of PPI network. The number of genes is plotted as a function of their degree reflecting a power-law like distribution. The red line corresponds to a power-law distribution. (B) Sub-network of candidate genes. The large-sized black-colored nodes represent the candidate genes (ABCC1, ABCG2, CERK, MMP-2, MMP-9, and SPHK1), while the small gray color nodes represent the corresponding interactor genes. Interactions are represented in gray color and the depth of color represents the strength of correlations. (C) Shortest path lengths among candidate genes. Heatmap of shortest path length among the candidate genes, where the values represent the number of shortest paths between any pair.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques:

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 9. (A–D) Gene expression of drug transporters in breast cancer patients (A) ABCC1, (B) ABCG2 in local cohort (C) ABCC1, (D) ABCG2 in TCGA cohort and (E–G) Protein expression of drug transporters (E) Representative blots in adjacent normal (N) and tumor (T) tissues, (F) Densitometric analysis of ABCG2 and (G) ABCC1 levels in adjacent normal and tumor tissues.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques: Gene Expression, Expressing

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 10. Correlation of CERK with (A) ABCC1, (B) ABCG2 and SPHK1 with (C) ABCC1, (D) ABCG2 in local cohort and CERK with (E) ABCC1, (F) ABCG2 and SPHK1 with (G) ABCC1, (H) ABCG2 in TCGA cohort.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques:

Journal: ACS pharmacology & translational science

Article Title: All Blood Brain Barrier Cell Types Demonstrate Capability to Influence Differential Tenofovir and Emtricitabine Metabolism and Transport in the Brain.

doi: 10.1021/acsptsci.4c00510

Figure Lengend Snippet: Figure 3. TFV/FTC efflux transporters and nucleotide-metabolizing kinases are differentially active in BBB cells. Brain microvascular endothelial cell, pericyte, and astrocyte monocultures were loaded with dyes for (A) P-gp (rhodamine 123, 10 μM), (B) MRP4 (monobromobimane, 10 μM), and (C) BCRP (Hoechst 33342, 5 μg/mL) for 15 min at 37 °C, 5% CO2. The dyes were allowed to efflux out for 2 h at 37 °C and 5% CO2, after which flow cytometry was performed to quantify intracellular fluorescence (mean fluorescence intensity, MFI) as an indicator of efflux capacity. Statistical analysis was performed using one-way ANOVA. (D) P-gp and (E) BCRP expressions were confirmed by Western blot, as these proteins were not reliably measurable by proteomics analyses. (F) Endogenous PK and (H) TK activities were measured in primary human BBB cell monocultures by a colorimetric activity assay and DiviTum activity assay, respectively. Four to eight independent experiments (represented by individual dots) were performed. Statistical analysis was performed using a Brown−Forsythe and Welch ANOVA test. (G) TFV-DP formation in human primary BBB cell lysates was measured by a CKB-mediated TFV metabolism assay using LC−MS/MS. Cell lysates were incubated with TFV-monophosphate (TFV-MP) and phosphocreatine (+CKB Substrates) or a mixture of phosphocreatine, phosphoenolpyruvate, and ATP (+Phosphate Donors) for 30 min at 37 °C. The reaction was quenched by LC−MS-grade methanol, and peak area of TFV-DP was measured by LC−MS/MS. Three to five independent experiments were performed (represented by individual dots). Statistical analysis was performed by using one-way ANOVA by GraphPad software. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Western blots were performed as previously described.16 Blots were probed with antibodies with specificity to BCRP (NBP2-22124, Novus Bio, Centennial, CO), P-gp (PA5-61300, Invitrogen), MRP4 (12705, Cell Signaling Technology), MRP1 (Ab24102, Abcam), ENT1 (PA5-116451, Invitrogen), PGK1 (PA528612, Invitrogen), AK2 (11014-1-AP, Proteintech, Rosemont, IL), CKB/CKM (15137-1-AP, Proteintech), TK (15691-1-AP, Proteintech), CMPK (11360-1-AP, Proteintech), DCK (17758-1-AP, Proteintech), PKM1 (15821-1-AP, Proteintech), and PKLR (2456-1-AP, Proteintech), overnight at 4 °C, washed with TBS-T, and probed with the appropriate secondary antibody (ab97023 and Ab97051, Abcam) for 1 h at room temperature.

Techniques: Flow Cytometry, Fluorescence, Western Blot, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Incubation, Software

Journal: Journal of Biological Chemistry

Article Title: Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

doi: 10.1074/jbc.m109.090506

Figure Lengend Snippet: FIGURE 2. Comparison of GSH transport-related MRPs and ABCG2 mRNA expression in epithelial MDA1586, A549, H1975, H460, HN4, and H157 cancer cells. Relative basal mRNA expression of the five known MRPs shown to transport GSH are compared with ABCG2 in epithelial cancer cells. The expression levels of ABCG2 best correspond to 25-DHC-induced GSH efflux and follow the order of 1586 A549 H1975 H460 HN4 H157. Values were normalized to a glyceraldehyde-3-phosphate dehydrogenase standard curve. The pattern of GSH efflux positively corresponds with ABCG2 expression in the cells. BCRP, breast cancer resistance protein; CFTR, cystic fibrosis transmembrane conductance regulator.

Article Snippet: A and B, HN4 cells were transfected with the OriGene ABCG2 transfection-ready plasmid, which resulted in a 2-fold increase in ABCG2 protein expression.

Techniques: Comparison, Expressing

Journal: Journal of Biological Chemistry

Article Title: Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

doi: 10.1074/jbc.m109.090506

Figure Lengend Snippet: FIGURE 3. Elevated levels of extracellular GSH in ABCG2-expressing yeast. Yeast expressing human ABCG2 protein (filled bars) for 24 h had 2.5- fold higher extracellular GSH compared with yeast not expressing the ABCG2 protein (open bars). Data are presented as the mean S.E. (error bars), and columns with nonidentical superscripts are significantly different (p 0.05) as determined using ANOVA with the Tukey post comparison test.

Article Snippet: A and B, HN4 cells were transfected with the OriGene ABCG2 transfection-ready plasmid, which resulted in a 2-fold increase in ABCG2 protein expression.

Techniques: Expressing, Comparison

Journal: Journal of Biological Chemistry

Article Title: Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

doi: 10.1074/jbc.m109.090506

Figure Lengend Snippet: FIGURE 4. MTX and 2,5-DHC alter extracellular GSH levels in ABCG2- expressing yeast. A, the ABCG2 substrate MTX decreased extracellular GSH levels in human ABCG2-expressing yeast (open bars) but not in the vector controls (filled bars). B, 2,5-DHC treatments (5 M and 10 M) significantly increased extracellular GSH levels in ABCG2-expressing yeast (open bars) but not in the vector controls (filled bars). Data are presented as the mean S.E. (error bars), and columns with nonidentical superscripts are significantly differ- ent (p 0.05) as determined using ANOVA with the Tukey post comparison test.

Article Snippet: A and B, HN4 cells were transfected with the OriGene ABCG2 transfection-ready plasmid, which resulted in a 2-fold increase in ABCG2 protein expression.

Techniques: Expressing, Plasmid Preparation, Comparison

Journal: Journal of Biological Chemistry

Article Title: Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

doi: 10.1074/jbc.m109.090506

Figure Lengend Snippet: FIGURE 5. Overexpressing human ABCG2 in epithelial head and neck cancer HN4 cells increases extra- cellular GSH levels. A and B, HN4 cells were transfected with the OriGene ABCG2 transfection-ready plasmid, which resulted in a 2-fold increase in ABCG2 protein expression. C and D, the increase in ABCG2 protein expression resulted in 2.5-fold increase in extracellular GSH levels over vector control (C) and no significant changes in intracellular GSH levels over 48 h (D). Data are presented as the mean S.E. (error bars), and statistical significance was determined using Student’s t test.

Article Snippet: A and B, HN4 cells were transfected with the OriGene ABCG2 transfection-ready plasmid, which resulted in a 2-fold increase in ABCG2 protein expression.

Techniques: Transfection, Plasmid Preparation, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

doi: 10.1074/jbc.m109.090506

Figure Lengend Snippet: FIGURE 6. Silencing ABCG2 in epithelial head and neck cancer MDA1586 cells decreased extracellular GSH levels. MDA1586 cells were transfected with a shRNA plasmid targeting ABCG2. A and B, silencing ABCG2 resulted in a 2.6-fold decrease in ABCG2 protein expression compared with the vector control. C and D, the loss of ABCG2 protein expression correlated with a 2.7-fold decrease in extracellular GSH levels compared with vector control (C) and no significant changes in intracellular GSH levels (D) over 48 h. Data are presented as the mean S.E. (error bars), and statistical significance was determined using Student’s t test.

Article Snippet: A and B, HN4 cells were transfected with the OriGene ABCG2 transfection-ready plasmid, which resulted in a 2-fold increase in ABCG2 protein expression.

Techniques: Transfection, shRNA, Plasmid Preparation, Expressing, Control