Figure 4. A39 is stabilized through a coordinated sensing of mitochondrial iron-sulfur cluster by the matrix cysteines. A39 regulation occurs in neurons.

(A) Western blot of A39 showing BSO treatment (1mM, 2d) increased A39 in the control K562 cells but did not affect NDUFA9 level, while the basal level of A39 and NDUFA9 were increased in the AFG3L2 CRISPR KO K562 cells.

(B) Diagram of iron manipulation strategies.

(C) Western blot of A39 in K562 cells treated with either or a combination of BSO (1mM, 2d) and DFO (20 μM, 2d).

(D) Western blot of A39 with and without BSO (1mM, 2d) in the CRISPR KO K562 cells of either or both mitochondrial iron transporters SLC25A28 and SLC25A37.

(E) Western blot of A39 upon BSO (1mM) treatment for 1 and 2d in the CRISPR KO K562 cells of HSCB or GLRX5.

(F) Diagram of A39 cysteine labeling and mobility shift assay. Cells were treated with either or a combination of BSO (100 μM, 2d) and DFO (deferoxamine, 20 μM, 2d).

(G) Anti-FLAG western blot and mobility shift assay for A39-FLAG in the indicated conditions, without or with cysteine labeling.

(H) Diagram of neuronal differentiation from the iPSC line, KOLF2.1J cells expressing dox-inducible NGN2 in the safe locus.

(I) Western blot of A39, HSCB, AFG3L2, and Synaptophysin, a synaptic marker from the indicated days (H) during neuronal differentiation. GAPDH, loading control.

(J) Western blot of A39 in the mature iNeurons (differentiation Day 21) upon 1 mM BSO treatment for 3 and 7 d. GAPDH, loading control.

(K) Western blot of A39 and IRP2 in the mature iNeurons (differentiation Day 21), treated with either or a combination of BSO (1mM, 7d) and DFO (20 μM, 7d). GAPDH, loading control.

(L) A39 regulation model.

See also Figure S3.