Journal: Frontiers in pharmacology

Article Title: Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

doi: 10.3389/fphar.2023.1331687

Figure Lengend Snippet: FIGURE 2 ABCG1 is upregulated in the MTX resistance cell line Reh-MTXR. (A) Illustration of the GSEA of upregulated ABC transporters pathway in Reh-MTXR cells. FDR <0.25 were defined as the significantly enriched gene sets. (B) RNA-seq analysis of differentially expressed genes upregulated and downregulated more than two folds in Reh cells and Reh-MTXR cells. Red dots represent upregulated genes, and blue dots represent downregulated genes. (C) mRNA levels of FPGS, GGH, DHFR, RFC1, ABCG1, ABCG2, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCB1 genes in Reh and Reh- MTXR cells were analyzed by RT-qPCR. Expression levels were normalized to a reference mRNA of Actin. Expression of genes in Reh was set as 1. Data are presented as mean ± SD of three independent tests, ****p < 0.0001, two-tailed Student’s t-test. (D) Cellular ABCG1 protein levels were detected by Western blot. Expression of Actin was measured as a loading control.

Article Snippet: The primary antibodies included β-actin (66009-1-Ig, Proteintech, Wuhan, China, 1:8000 dilution), ABCG1 (13578-1-AP, Proteintech, Wuhan, China, 1:5000 dilution), FPGS (ab184564, Abcam, Cambridge, United Kingdom, 1:1000 dilution), GGH (13264-1-AP, Proteintech, Wuhan, China, 1:2000 dilution).

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Control

Journal: Frontiers in pharmacology

Article Title: Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

doi: 10.3389/fphar.2023.1331687

Figure Lengend Snippet: FIGURE 4 ABCG1 promotes MTX efflux in Reh-MTXR cells. (A, B) Reh, Reh-MTXR, and Reh-MTXR ABCG1 knockdown cells were exposed to 2 μM MTX, and after 24 h of accumulation of MTX, the levels of MTX polyglutamated metabolites were determined by LC-MS. (A) Diagram used for identifying MTX polyglutamated metabolites by LC-MS in various Reh cells. (B) Relative levels of MTX, MTX-Glu2, MTX-Glu3, and MTX-Glu4 in various Reh cells, each group was normalized to the Reh group, which was set as 100%. The experiment was performed in triplicates, and data are presented as mean ± SD. ***p < 0.001, ****p < 0.0001, two-tailed Student’s t-test. (C) Western blot analyses of FPGS and GGH in Reh, Reh-MTXR, and Reh-MTXR ABCG1 knockdown cells. We measured the expression of Actin as a loading control. (D) After 20 min of incubation in the medium containing 10 μM MTX, we washed the Reh, Reh-MTXR, and Reh-MTXR ABCG1 knockdown cells and incubated them in the drug-free medium. Efflux was measured by determining the level of MTX remaining in the cells after 0, 5, 15, and 30 min of incubation in the drug-free medium. The data are represented as the relative amount of MTX remaining in cells and are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t-test, Reh-MTXR group compared with Reh group; MTXR-shABCG1 group compared with Reh-MTXR group.

Article Snippet: The primary antibodies included β-actin (66009-1-Ig, Proteintech, Wuhan, China, 1:8000 dilution), ABCG1 (13578-1-AP, Proteintech, Wuhan, China, 1:5000 dilution), FPGS (ab184564, Abcam, Cambridge, United Kingdom, 1:1000 dilution), GGH (13264-1-AP, Proteintech, Wuhan, China, 1:2000 dilution).

Techniques: Knockdown, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Western Blot, Expressing, Control, Incubation

Journal: BMC Complementary and Alternative Medicine

Article Title: Serum protein gamma-glutamyl hydrolase, Ig gamma-3 chain C region, and haptoglobin are associated with the syndromes of pulmonary tuberculosis in traditional Chinese medicine

doi: 10.1186/s12906-015-0686-4

Figure Lengend Snippet: Analysis of differentially expressed proteins by ELISA and one-way ANOVA. a GGH; b IGHG3; c HPT. PYD: pulmonary Yin deficiency syndrome; HFYD: hyperactivity of fire due to Yin deficiency syndrome; DQY: deficiency of Qi and Yin syndrome; GGH: gamma-glutamyl hydrolase; IGHG3: Ig gamma-3 chain C region; HPT: haptoglobin. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human Haptoglobin ELISA kit (Abcam, London, England; the dilution was 1:2000), human IGHG3 ELISA kit (CUSABIO Biotech, Wuhan, Hubei, China; the dilution factor was 1:5000), and human GGH ELISA kit (CUSABIO Biotech, Wuhan, Hubei, China; the sample dilution was 1) were used to perform experiment in duplicates in accordance with the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Oncology Reports

Article Title: High pemetrexed sensitivity of docetaxel-resistant A549 cells is mediated by TP53 status and downregulated thymidylate synthase

doi: 10.3892/or.2017.5951

Figure Lengend Snippet: Characterization of the effects of TS and GGH expression on PEM sensitivities. The expression levels of folate synthesis enzymes were examined by western blot analysis in DOC-resistant A549 sublines. The enzymes of TS and DHFR are associated with de novo synthesis, and TK1 is associated with the salvage pathway of DNA synthesis. FPGS and GGH are poly/mono-glutamylation counteracting enzymes. Anti-β-actin was used as a control for loading. Representative data are shown as follows: (A) overexpression of TS in A549/D16 was detected using (B) western blotting with PEM sensitivities determined by (C) MTT assay. Similar procedures were performed with (D) GGH overexpression and the (E) effect on PEM sensitivity. TS, thymidylate synthase; GGH, γ-glutamyl hydrolase; PEM, pemetrexed; DOC, docetaxel; DHFR, dihydrofolate reductase; FPGS, folylpolyglutamate synthase.

Article Snippet: Proteins (10–30 μg) transferred onto polyvinylidene fluoride (PVDF) membranes were reacted with polyclonal anti-TS (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-DHFR, FPGS, GGH and TK1 (GeneTex, Irvine, CA, USA), anti-β-actin (NeoMarker, Fremont, CA, USA) and anti-TP53 (Dako, Carpinteria, CA, USA) separately, followed by conjugation of anti-rabbit (Santa Cruz Biotechnology, Inc.) or anti-mouse (Calbiochem, La Jolla, CA, USA) IgG to horseradish peroxidase.

Techniques: Expressing, Western Blot, DNA Synthesis, Control, Over Expression, MTT Assay