Figure 5.
Matrix Detachment Leads to Cluster Formation, Hypoxia Induction, and Hif1α-Mediated Mitophagy
(A) Representative microscopy images of detached 293T, HeLa, and A549 cells forming clusters. Scale bars represent 100 μm.
(B) Western blot analysis of pan-cadherin and β-actin levels of attached and detached 293T, HeLa, and A549 cells. Equal protein levels were loaded per condition.
(C) Image-iT green hypoxia reagent staining of detached 293T cells cultured in static (left panel) and shaken (right panel) conditions. Cells were cultured in detached conditions for 3 days before live cell staining was performed.
(D) Western blot analysis of attached and detached 293T, HeLa, and A549 cells showing expression of HIF1α, NIX, BNIP3, and β-actin (NIX was detected on a duplicate blot). Equal protein amounts were loaded per condition and verified by ponceau staining. β-Actin was used to verify the integrity of the samples.
(E) Western blot showing expression of HIF1α, NIX, BNIP3, and β-actin (NIX was detected on a duplicate blot) in 293T attached cells treated with CoCl2 in the presence or absence of siHif1α. Equal protein amounts were loaded per condition and verified by ponceau staining. β-Actin was used to verify the integrity of the samples.
(F) Western blot analysis of detached 293T, HeLa, and A549 cells showing expression of HIF1α, NIX, BNIP3 and β-actin (NIX was detected on a duplicate blot) in the absence or presence of siHIF1. Equal protein amounts were loaded per condition and verified by ponceau staining. β-Actin was used to verify the integrity of the samples.
(G) HeLa cells were transfected with mitochondrial-targeted mKeima and cultured in attached or detached conditions for 3 days before flow cytometry analysis. Mitochondria in cytosol (pH 7) are represented by mKeima with excitation of 405 nm, while mitochondria in lysosomes (pH 4) are represented by mKeima with excitation of 561 nm. Mitophagy is reflected by the percentage of cells in the upper panel represented by the graph showing mean ± SD of triplicate cultures in a representative experiment.
(H) Mitochondrial mass of 293T and HeLa attached and detached cells were measured using Mito Tracker green fluorescent dye and analyzed by flow cytometry.
(I) Western blot analysis of MiaPACA, A549, SW480, MDA-MB-468, HeLa and 293T attached and detached cells showing expression of Tom20 and β-actin. Actin was detected on a duplicate blot. Equal protein amounts were loaded per condition and verified by ponceau staining. β-Actin was used to verify the integrity of the samples.
(J) Mitochondrial-targeted mkeima-expressing HeLa cells were cultured in detached conditions in the presence of non-targeting, Hif1α or BNIP3 + NIX siRNA and analyzed by flow cytometry for mitophagy as in (F).
(K) Mitochondrial mass of HeLa cells cultured in detached conditions in the presence of non-targeting, Hif1α or BNIP3 + NIX siRNA. Mitochondrial mass was measured using NAO staining and flow cytometry analysis.
(L) Mitochondrial ROS of HeLa cells cultured in detached conditions in the presence of non-targeting, Hif1α or BNIP3 + NIX siRNA measured by MitoSOX Red staining and flow cytometry analysis.
(G, H, J, K, and L) Data are presented as mean ± SD of triplicate cultures of representative experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗∗p ≤ 0.0001, unpaired t test.