Fig. 2: TNTs between BMSCs transfer JJN3 mitochondria in post-fission state and contribute to JJN3 resistance to OGD. | Blood Cancer Journal

Fig. 2: TNTs between BMSCs transfer JJN3 mitochondria in post-fission state and contribute to JJN3 resistance to OGD.

From: Tunneling nanotubes between bone marrow stromal cells support transmitophagy and resistance to apoptosis in myeloma

Fig. 2

A Schematic representation of the co-culture model used here. Mitochondria of JJN3 cells were stained with fixable Mitotracker Deep Red and co-cultured under normoxic (Nx) or OGD conditions with BMSCs. The co-culture was analyzed for mitochondrial transfer and colocalization with Lamp1 in BMSCs through confocal microscopy. B Quantification of mitochondrial transfer from JJN3 cells to BMSCs under Nx and OGD conditions. OGD triggers intercellular mitochondrial transfer for small post-fission mitochondria. ****p < 0.0001; **p = 0.0082. C JJN3 cells interact with BMSCs through CX43. Confocal microscopy of the OGD co-culture shows high CX43 abundance in the interaction region, and mitochondria frequently colocalize with CX43. D OGD triggers the colocalization of JJN3 mitochondria and Lamp1 in BMSCs. Note the presence of an actin cage typical of damaged mitochondria destined for mitophagy (central panel). Once inside BMSCs, multiple fission points were observed in JJN3 mitochondria (bottom panel, white arrows). **p = 0.0079. E TNTs between BMSCs in co-culture with JJN3 Mito under OGD conditions contain lysosomes and small JJN3 mitochondria in post-fission state. The co-culture was analyzed using high-resolution confocal microscopy for F-actin (cyan), JJN3 mitochondria (red), and Lamp1 (green). Note the presence of small mitochondria, with an average size of 1 ± 0.5 µm, originating from JJN3 cells inside TNTs between BMSCs. Lamp1 frequently co-localizes with JJN3 mitochondria inside the TNTs. F TNTs between BMSCs can transfer small JJN3 mitochondria in a post-fission state from one BMSC to another. A co-culture in OGD conditions, composed of donor BMSC (JJN3 Mito) (red) and acceptor BMSCs stained with DiI (Cyan), shows F-actin-positive (green) TNTs connecting donor and acceptor BMSCs (inset a) and the presence of JJN3 mitochondria inside the acceptor BMSCs (inset a). G Cytochalasin-D (CytoD) in OGD destroys TNTs between BMSCs. ****p < 0.0001. H TNTs between BMSCs contribute to JJN3 apoptotic resistance in OGD. Analysis of caspase 3/7 activity (green) reveals that pre-treatment of BMSC with CytoD prevents the rescue of JJN3 cells in OGD conditions. ***p = 0.0003; *p = 0.04 (OGD vs OGD+BMSCs); *p = 0.015 (OGD+BMSCs+DMSO vs OGD+BMSCs+CytoD). I Model of TNTs-mediated BMSCs-to-BMSCs JJN3 mitochondrial transfer supporting JJN3-to-BMSCs transmitophagy.

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