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. 2023 Oct 11;26(11):108168. doi: 10.1016/j.isci.2023.108168

Figure 3.

Figure 3

MUC1-C is necessary for induction of GLUT1 and HK2 in TNBC CSCs

(A) Lysates from 2D and S1-S3 cells were immunoblotted with antibodies against the indicated proteins.

(B) Lysates from BT-549/tet-MUC1shRNA S6 3D cells treated with vehicle or DOX for 10 days (left) and BT-549 cells treated with vehicle or 2.5 μM GO-203 for 2 days (right) were immunoblotted with antibodies against the indicated proteins.

(C) GSEA of genes in (i) BT-549/tet-MUC1shRNA S6 cells treated with DOX vs. vehicle and (ii) BT-549 cells treated with GO-203 vs. vehicle using the HALLMARK_MYC_TARGETS_V1 gene signature.

(D) Lysates from BT-549/tet-MYCshRNA S6 3D cells treated with vehicle or DOX for 10 days were immunoblotted with antibodies against the indicated proteins.

(E) Nuclear lysates from BT-549 2D and 3D cells were immunoprecipitated with anti-MUC1-C or a control IgG (left). Nuclear lysates from BT-549 3D cells treated with vehicle or 2.5 μM GO-203 for 3 days were immunoprecipitated with anti-MUC1-C or a control IgG (right). The precipitates were immunoblotted with antibodies against the indicated proteins.

(F) Schema of GLUT1 with positioning of the PLS, pELS, and dELS regions. Soluble chromatin from BT-549 2D and 3D cells was precipitated with a control IgG or anti-MYC antibody. The DNA samples were amplified by qPCR with primers for the GLUT1 PLS, pELS, and dELS regions (Table S2). The results (mean ± SD of 3 determinations) are expressed as fold enrichment relative to that obtained with the IgG control (assigned a value of 1). Asterisks represent ∗p ≤ 0.05.

(G) Schema of HK2 with positioning of the PLS, dELS1, and dELS2 regions. Soluble chromatin from BT-549 2D and 3D cells was precipitated with a control IgG or anti-MYC antibody. The DNA samples were amplified by qPCR with primers for the HK2 PLS, dELS1, and dELS2 regions (Table S2). The results (mean ± SD of 3 determinations) are expressed as fold enrichment relative to that obtained with the IgG control (assigned a value of 1). Asterisks represent ∗∗p ≤ 0.01.

(H and I) Soluble chromatin from BT-549/tet-MUC1shRNA 3D cells treated with vehicle or DOX was precipitated with a control IgG or anti-MYC antibody. The DNA samples were amplified by qPCR with primers for the GLUT1 PLS and pELS regions (H), and HK2 PLS region (I). The results (mean ± SD of 3 determinations) are expressed as fold enrichment relative to that obtained with the IgG control (assigned a value of 1). Asterisks represent ∗p ≤ 0.05, ∗∗p ≤ 0.01.

(J) Glucose uptake was measured in BT-549 3D cells treated with vehicle or 2.5 μM GO-203 for 3 days. The results (mean ± SD of 3 determinations) are expressed as luminescence (relative light unit, RLU). Asterisks represent ∗∗∗p ≤ 0.001.

(K) BT-549 3D cells were treated with vehicle or 5 μM 2DG for 7 days. The relative SFE is expressed as the mean ± SD of three determinations as compared to that obtained for vehicle-treated cells (assigned a value of 1). Asterisks represent ∗p ≤ 0.05.