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

Figure 6.

Figure 6

MUC1-C suppresses superoxide production in CSCs

(A) BT-549/tet-MUC1shRNA mammospheres treated with vehicle or DOX for 10 days were stained with MitoSOX Red. Shown are representative fluorescence microscopy images.

(B and C) BT-549/tet-MUC1shRNA (B) and MDA-MB-436/tet-MUC1shRNA (C) mammospheres were treated with vehicle or DOX for 10 days. MitoSOX Red flow cytometry data (mean ± SD of 3 determinations) are expressed as relative geometric mean fluorescence intensity (gMFI) compared to that obtained for vehicle-treated cells (assigned a value of 1)(left). Trypan blue staining results (mean ± SD of 3 determinations are expressed as the % cell death (right). (C) MitoSOX Red flow cytometry data in MDA-MB-436/tet-MUC1shRNA mammospheres treated with vehicle or DOX for 10 days. The results (mean ± SD of 3 determinations) are expressed as fold-change of gMFI for the mammosphere cells (left). MDA-MB-436/tet-MUC1shRNA mammospheres treated with vehicle or DOX for 10 days were monitored for cell death by trypan blue staining. The results are expressed as the % cell death (mean ± SD of 3 determinations)(right). Asterisks represent ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

(D) BT-549 mammospheres treated with vehicle or 5 μM GO-203 for 3 days were stained with MitoSOX Red. Shown are representative fluorescence microscopy images.

(E and F) BT-549 (E) and MDA-MB-436 (F) mammospheres were treated with vehicle or 5 μM GO-203 for 3 days. MitoSOX Red flow cytometry data (mean ± SD of 3 determinations) are expressed as relative geometric mean fluorescence intensity (gMFI) compared to that obtained for vehicle-treated cells (assigned a value of 1). The results (mean ± SD of 3 determinations) are expressed as fold-change of gMFI for the mammophere cells. Asterisks represent ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

(G) BT-549 mammospheres treated with vehicle or the indicated GO-203 concentrations for 3 days were stained with JC-1. Fluorescence images are shown for the control and GO-203-treated cells (upper panels). The JC-1 stained mammosphere cells were analyzed by flow cytometry for assessment of JC-1 red vs. green emission as a measure of the MMP (lower panels). The results (mean ± SD of 3 determinations) are expressed as the % depolarized mitochondria (JC-1 monomers). Asterisks represent ∗∗p ≤ 0.01.

(H) MDA-MB-436 mammospheres treated with vehicle or the indicated GO-203 concentrations for 3 days were stained with JC-1. The JC-1 stained mammosphere cells were analyzed by flow cytometry for assessment of JC-1 red vs. green emission. The results (mean ± SD of 3 determinations) are expressed as the % depolarized mitochondria (JC-1 monomers). Asterisks represent ∗∗∗∗p ≤ 0.0001.

(I and J) BT-549 (I) and MDA-MB-436 (J) mammospheres treated with vehicle or 5 μM GO-203 for 3 days were monitored for cell death by trypan blue staining. The results (mean ± SD of 3 determinations) are expressed as the % cell death. Asterisks represent ∗∗∗∗p ≤ 0.0001.