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. 2022 Sep 12;41(42):4709–4723. doi: 10.1038/s41388-022-02460-1

Fig. 2. ISCA2 is the molecular target of HIF-α inhibitors.

Fig. 2

A Schematic showing sample preparation workflow for DARTS assay. Lysates from ACHN cells that were exposed to 1% O2 for 16 h were divided into 4 equal samples and subjected to indicated treatments. B (i) Mass spectrometry data from DARTS assay indicating intensity of ISCA2 fragments in cell lysates treated with Pronase together with #1 or DMSO (Con); (ii) Western blot showing effects of control or Pronase treatment in ACHN cell lysates treated with indicated concentrations of #1. Densitometric analysis of ISCA2 band intensities normalized to actin is shown above blots. B, C Thermal shift assays showing effects of incubation of recombinant ISCA2 (4 μg) with (B) DMSO (green) or 200 μM #1 (magenta), or (C) DMSO (green) or 100 µM #25 (red). Quantitation of ISCA2 melting temperatures are shown inset (mean ± SEM). D Effects of transfections with non-targeting (Con) or ISCA2 (A2) siRNA on indicated proteins in hypoxic ACHN cells. Knockdown was performed using two consecutive siRNA transfections over 72 h each. E–G Iron content of 786-0 cells treated with (E) #1, (F) #25, for 24 h; or (G) transfected with siCon or siISCA2 (6 days) as determined using ICP-MS.