Additional file 1 of Hyperglycemia and O-GlcNAc transferase activity drive a cancer stem cell pathway in triple-negative breast cancer
Creators: name: Ayodeji, Saheed A.
name: Bao, Bin
name: Teslow, Emily A.
name: Polin, Lisa A.
name: Dyson, Greg
name: Bollig-Fischer, Aliccia
name: Fehl, Charlie
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Online Resource 1. Expanded TCGARNA-seq patient sample analysis for CSC pathway genes. a) O-GlcNAcaselevels between subtypes of breast cancer and normal tissue. b–d Pathway protein data and analysis, re-analyzed from data first analyzed andreported in our TET1 study.1 Non-tumor samples, N= 8; TNBC/basal-like samples, N = 81; HER2-enriched, N = 53; luminal A, N =208; luminal B, N = 110. Online Resource 2. Alternative confirmation that TET1 isO-GlcNAc modified. a Overexpression of Flag-HA-tagged TET1 catalytic domain in HEK293T and HEK293 cells. Stars indicate TET1-specific bands. b HEK293T cells were fed the azide-labeled per-acetyl galactosamineas a metabolic reporter of O-GlcNAc. Click chemistry was performed with biotinalkyne. HA immunoprecipitation was used to enrich Flag-HA-TET1. Streptavidinblot was used to visualize biotinylated samples. Online Resource 3. Comparison of OGT mRNA levels by qRT-PCRbetween high and low glucose conditions. Media containing high glucosewas added for 24 h following growth in low glucose media. Barsindicate variability across 3 technical replicates for each cell line. Data wascollected for 1 biological replicate per cell line, no significance wasdetermined. OnlineResource 4. SpliceAid diagram of TARDBP binding siteon OGT mRNA. Made using human OGT sequence andhttp://www.introni.it/splicing.html.Online Resource 5a. Gating strategy for analysis of cancer stem-like cell markers. Unstained controls for each condition were used to set the analysis parameters. Front scatter plotand side scatter plotwere use by areaor heightto determine cell morphology. CD44 was analyzed in the PE-Cy7 channel, CD133 was analyzed in the APC channel, and EpCam was analyzedin the Y780 channel to set background. The text below each plot indicates the sample type, then the gating strategy, and the number indicates the total number of events for each run. Cell lines: GFP-vector-expressing MDA-MB-468 cells or MBD2_V2-overexpressing MDA-MB-468 cell lines. Online Resource 5b. Analysis of cancer stem-like cell markers CD44, CD133, and EpCam in bulk TNBC cells. Cell lines: stable GFP-vector-expressing MDA-MB-468 cells were treated with DMSO control. Cells were first gated by CD44, then analyzed for CD133 and EpCam staining. All three markers represent cancer stem-like cells. Results presented for three biological replicates. Online Resource 5c. Analysis of cancer stem-like cell markers CD44, CD133, and EpCam in bulk TNBC cells. Cell lines: stable GFP-vector-expressing MDA-MB-468 cells were treated with OGT inhibitor. Cells were first gated by CD44, then analyzed for CD133 and EpCam staining. All three markers represent cancer stem-like cells. Results presented for three biological replicates. Online Resource 5d. Analysis of cancer stem-like cell markers CD44, CD133,and EpCam in bulk TNBC cells. Cell lines: stable MBD2v2-overexpressingMDA-MB-468 cells were treated with DMSO control. Cells were first gated byCD44, then analyzed for CD133 and EpCam staining. All three markers representcancer stem-like cells. Results presented for three biological replicates. Online Resource 5e. Analysis of cancer stem-like cell markers CD44, CD133,and EpCam in bulk TNBC cells. Cell lines: stable MBD2v2-overexpressing MDA-MB-468cells were treated with OGT inhibitor. Cells were firstgated by CD44, then analyzed for CD133 and EpCam staining. All three markersrepresent cancer stem-like cells. Results presented for three biologicalreplicates. Online Resource 5f. Analysis of cancer stem-like cell markers CD44, CD133,and EpCam in bulk TNBC cells. Bars indicate the standard error of the mean ofthree biological replicates. Statistical analysis: * indicates P ≤ 0.05, **indicates P ≤ 0.01. Online Resource 6. Weight of mice onhigh fat dietrelative to control diet. The difference was significant at 5 weeks, P &lt; 0.001. 12 mice per group.