Fig 2: Inhibition of and/or knocking-out HIF-1α or miR-210 abolished Nano-Ni-induced Rad52 down-regulation. A–C BEAS-2B cells were pretreated with 1 µM of 17-AAG for 4 h, followed by treatment with 20 µg/mL of Nano-Ni for 24 h. D–F HIF-1α wild-type (+ / +) and knock-out (−/−) cells were treated with 20 µg/mL of Nano-Ni for 24 h. G–I BEAS-2B cells were transfected with mirVana™ miRNA inhibitor for has-miR-210-3p or Negative Control #1 for 24 h, followed by treatment with 20 µg/mL of Nano-Ni for another 24 h. A, D, G miR-210 expression was determined by real-time PCR. Values of miR-210 expression was normalized to the endogenous control U6 snRNA. Data are shown as mean ± SEM (n = 3–4). B–C, E–F, H–I Nuclear proteins were subjected to Western blot. Equal nuclear protein loading was verified by Coomassie Brilliant Blue staining. B, E, H are results of a single Western blot experiment, while C, F, I are quantified band densitometry readings averaged from at least 3 independent experiments ± SEM of Western blot results. *, p < 0.05 vs. control; #, p < 0.05 vs. group with Nano-Ni treatment, but without 17-AAG treatment (A, C), Nano-Ni-treated HIF-1α (+ / +) group (D, F), or group with Negative Control transfection and Nano-Ni treatment (G, I)

Fig 3: Long-term Nano-Ni exposure induced cell transformation, which was attenuated by overexpression of Rad52. A BEAS-2B cells were transduced with lentiviral particles containing human Rad52 ORF as described in the Methods. 11 puromycin-resistant colonies were picked and expanded, and the total protein from each colony was isolated for Western blot. The exposure time was 2 s. β-actin served as loading control. WT, wild-type. B, C The effects of Nano-Ni on anchorage-independent growth of cells by soft agar colony formation assay. Cells were exposed to 0, 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles as described in the Methods. The colonies were stained with INT/BCIP solution (B) and quantified using ImageJ software (C). Data are shown as mean ± SEM (n = 3 ~ 6). *, p < 0.05 vs. control; #, p < 0.05 vs. wild-type group with same dose of Nano-Ni treatment

Fig 4: Dysregulation of DNA damage response-associated proteins and the HIF-1α/miR-210/Rad52 signaling pathway in BEAS-2B cells after long-term Nano-Ni exposure. BEAS-2B cells were treated with 0, 0.25 and 0.5 µg/mL of Nano-Ni for 21 cycles as described in the Methods. A is the result of a single Western blot experiment. Nuclear protein was subjected to Western blot. Equal nuclear protein loading was verified by Coomassie Brilliant Blue staining. B, C are quantified band densitometry readings averaged from 3 independent experiments ± SEM of Western blot results. D miR-210 expression was determined by real-time PCR. Values of miR-210 expression was normalized to the endogenous control U6 snRNA. Data are shown as mean ± SEM (n = 3). *, p < 0.05 vs. control

Fig 5: Schematic diagram of the possible mechanisms involved in Nano-Ni-induced cell transformation. Nano-Ni exposure causes DNA damage, which induces the DNA damage response. Repeated insults may cause erroneous DNA repair. Nano-Ni exposure also induces HIF-1α nuclear accumulation, which causes defective DNA repair through up-regulation of miR-210 and down-regulation of Rad52. Both DNA damage and defective DNA repair may contribute to increased genomic instability, leading to cell transformation