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

Figure 4.

Figure 4

MUC1-C is necessary for expression of nuclear genes encoding components of the mitochondrial ETC

(A) GSEA of genes in BT-549 cells grown in 3D vs. 2D culture (left) and BT-549/tet-MUC1shRNA 3D cells treated with DOX vs. vehicle control (right) using the WP_ETC_OXPHOS_MITOCHONDRIA gene signature.

(B) WP_ETC_OXPHOS_SYSTEM IN MITOCHONDRIA signature heatmaps of ETC encoding genes in (i) BT-549 cells grown in 3D vs. 2D culture, (ii) BT-549/tet-MUC1shRNA 3D cells treated with DOX vs. vehicle, (iii) BT-549 3D cells treated with GO-203 vs. vehicle, and (iv) BT-549/tet-MYCshRNA 3D cells treated with DOX vs. vehicle. The row indicator shows gene origins, nuclear DNA (black) and mtDNA (yellow).

(C) Expression of the indicated nuclear genes in BT-549/tet-MUC1shRNA 3D cells treated with DOX vs. vehicle for 7 days was determined by qRT-PCR. The results (mean ± SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Asterisks represent ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

(D–F) BT-549 3D vs. 2D cells and BT-549/tet-MUC1shRNA 3D cells treated with DOX vs. vehicle for 7 days were analyzed for SDHD (D) and CYCS (E) expression. The qRT-PCR results (mean ± SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for 2D cells or vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (F). Asterisks represent ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

(G) Schema of SDHD with positioning of an E-box in the pELS region. 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 SDHD pELS region (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.

(H) Schema of CYCS with positioning of an E-box in the pELS region. 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 CYCS PLS and pELS 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.

(I and J) Soluble chromatin from BT-549/tet-MUC1shRNA 3D cells treated with vehicle or DOX for 5 days was precipitated with a control IgG or anti-MYC antibody. The DNA samples were amplified by qPCR with primers for the SDHD pELS (I) and HK2 pELS regions (J). 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 ≤ 0.001.