Journal: Cancer Cell International

Article Title: Hyperglycemia and O-GlcNAc transferase activity drive a cancer stem cell pathway in triple-negative breast cancer

doi: 10.1186/s12935-023-02942-6

Figure Lengend Snippet: OGT is upregulated in TNBC tumors and cell lines. a , b Whole genome expression analyses of human patient samples targeting OGT mRNA expression in TNBC, matched breast tissue, and other subtypes of breast cancer. Line equals median, means were compared. Non-tumor samples, N = 8; TNBC/basal-like samples, N = 81; HER2-enriched, N = 53; luminal A, N = 208; luminal B, N = 110. NS indicates no significance (p > 0.05). c , d qRT-PCR analysis of OGT and OGA in non-tumor breast (MCF 10 A), two hormone-expressing BC lines, and three TNBC cell lines. Bars indicate standard error of the mean (SEM) from 3 independent experiments. e Western blot analysis of OGT and OGA protein expression in non-tumor MCF 10 A and tumor lines, with hormone status indicated. f , g Densitometry analysis of OGT and OGA expression in 6 cell lines, displayed relative to the MCF10A OGT band and normalized to actin. N = 1. h Western blot analysis of total cell O-GlcNAc (CST O-GlcNAc MultiMAb antibody) in 6 breast cell lines. N = 1. i Densitometry for total O-GlcNAc levels in 6 breast cell lines. j Dose-response for total cellular O-GlcNAcylation with OSMI-4 in MDA-MB-468 cells, 48 h treatment. Western blot performed with anti-O-GlcNAc MultiMAb. Bars indicate SEM of 3 biological replicates, with 3 technical replicates for each cell line. k TARDBP mRNA analysis by qRT-PCR upon OGT inhibition with OSMI-4- (10 µM, 24 h in low glucose media). Data is normalized to vehicle-treated controls. l Hypothesis for OGT inhibition impact on the TET1-driven target gene TARDBP based on published TET1 pathway [ref. 13]. m Western blot analysis of TARDBP protein expression in OSMI-4 treated (10 µM, 48 h in low glucose (1.0 g/L) media) or control (DMSO) treated TNBC cell lines. N = 2. n Densitometry analysis of TARDBP expression with OSMI-4 treatment, displayed relative to the untreated cells and normalized to tubulin

Article Snippet: Antibodies used for Western blot are: Anti-OGT (Cell Signaling Technology (CST), #24083), Anti-O-GlcNAc CTD110.6 (CST, #9875S), Anti-O-GlcNAc MultiMAb (CST #82332) Anti-Actin (Sigma, #A3853), Anti-Tubulin (Invitrogen, #MA1-80017), Anti-TET1 (Abnova Taiwan, #H00080312), Anti-TARDBP (Abnova, #H000234350-M01).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: The Role of TRIP6, ABCC3 and CPS1 Expression in Resistance of Ovarian Cancer to Taxanes

doi: 10.3390/ijms23010073

Figure Lengend Snippet: ABCC3, CPS1, and TRIP6 expression in paclitaxel-resistant NCI/ADR-RES, SKOV-3/RES, and OVCAR-3/RES. ( A ) Bar graph showing relative expression of ABCC3, CPS1 and TRIP6 genes in paclitaxel-resistant ovarian cancer cell lines (technical triplicates). ( B ) Representative immunoblots of CPS1 in paclitaxel-resistant ovarian carcinoma cell lines. CPS1 silenced SKOV-3/RES cells or non-specific siRNA transfected SKOV-3/RES cells and human liver tissue were used as controls. ( C ) Representative immunoblot of ABCC3 in paclitaxel-resistant cell lines. ABCC3 silenced MCF-7/RES breast cancer cells or non-specific siRNA transfected MCF-7/PacR breast cancer cells and human liver tissue were used as controls. ( D ) Representative immunoblot of TRIP6 in paclitaxel-resistant ovarian carcinoma cell lines. β-ACTIN served as a loading control. The size of TRIP6 band was confirmed previously by us .

Article Snippet: Following primary antibodies were applied onto the membranes and incubated overnight at 4 °C: anti -TRIP6 (HPA052813) and anti -ACTIN (clone AC-40) (A3853) from Merck (Darmstadt, Germany), anti -CPS1 [EPR7493-3] (ab129076) from Abcam (Cambridge, UK) and anti-MRP3 (PA5-23653) from ThermoFisher.

Techniques: Expressing, Western Blot, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: The Role of TRIP6, ABCC3 and CPS1 Expression in Resistance of Ovarian Cancer to Taxanes

doi: 10.3390/ijms23010073

Figure Lengend Snippet: Significant differences in the mRNA levels of ( A ) CPS1 and ( B ) TRIP6 genes and ( C ) CPS1 and TRIP6 proteins in ovarian carcinoma mouse xenografts after the treatment with paclitaxel and novel SB-Ts in vivo. ( A , B ) Gene expression differences are shown as a mean of fold change (2 −∆∆CT ) ± SD, between the control group (Group I), group treated with 10 mg/kg paclitaxel (Group II), 9 mg/kg paclitaxel + 1 mg/kg SB-T-121605 (Group III), 7 mg/kg paclitaxel + 3 mg/kg SB-T-121605 (Group IV), 9 mg/kg paclitaxel + 1 mg/kg SB-T-121606 (Group V), and 7 mg/kg paclitaxel + 3 mg/kg SB-T-121606 (Group VI). Statistical analysis was performed by the two-tailed Student´s t -test * p < 0.05, ** p < 0.01, *** p < 0.001). ( C ) Representative immunoblot of CPS1, TRIP6, and β-ACTIN proteins in each group of mouse xenografts. Each group consisted of five mice.

Article Snippet: Following primary antibodies were applied onto the membranes and incubated overnight at 4 °C: anti -TRIP6 (HPA052813) and anti -ACTIN (clone AC-40) (A3853) from Merck (Darmstadt, Germany), anti -CPS1 [EPR7493-3] (ab129076) from Abcam (Cambridge, UK) and anti-MRP3 (PA5-23653) from ThermoFisher.

Techniques: In Vivo, Gene Expression, Control, Two Tailed Test, Western Blot