Supplementary Data

Material and Methods

Cell Viability Measured with ATP Assay

The viability of rMC-1 cells was measured with the ATP assay kit (CellTiter-Glo® Luminescent Cell Viability Assay, Cat. #G7570, Promega), following the manufacturer’s instructions. Briefly, the rMC-1 cells were seeded on opaque-walled 96-well plates at a density of 1.0 x 104 cells per well, and treated with ZnCl2 (125 μM) or ZnCl2 (125 μM) + EPO (40 U/mL) for 24 hours. The plate was equilibrated at room temperature for 30 minutes to ensure uniform temperature across plate during ATP assay. Then, 100 μL of CellTiter-Glo® reagent was added to each well and mixed with the cells for 2 minutes on an orbital shaker to induce cell lysis. The plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The luminescence intensity was recorded using multi-mode plate reader (BioTek Synergy 2). The cell viability was expressed as the percentage of the normal control (without treatment), which was defined as 100% for each experiment.

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts of rMC-1 cells were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Cat. #P0027, Beyotime Biotechnology, Shanghai, China). A consensus oligonucleotide for HIF-1α containing the HIF-1α binding site and the parallel mutant probe were purchased from Beyotime Biotechnology (as shown in Supplementary Fig. 2A). EMSA assay was carried out according to the manufacturer’s instructions. Briefly, the 3’ end of the wild-type probe was labeled with EMSA Probe Biotin Labeling Kit (Cat. #GS008, Beyotime Biotechnology, Shanghai, China). Then the labeled probes were incubated with nuclear extracts (16 μg) in binding buffer at 25 ℃ for 20 minutes in a final volume of 10 μL. To confirm the specificity of DNA-protein interaction, the competition reaction was conducted by the addition of 100-fold molar excess of unlabeled wild-type probe (cold probe) or mutant probe into the reaction systems. The DNA-protein complexes were separated by 6.5% nondenaturing polyacrylamide gel in 0.5X TBE buffer by electrophoresis and then transferred onto nylon membrane. Finally, the biotin-labeled complexes were detected using Chemiluminescent EMSA Kit (Cat. #GS009, Beyotime Biotechnology, Shanghai, China).

Supplementary Figures and Figure legends

Table 1 Primer information for zinc transporters

Gene / Accession No. / Forward / Reverse / Product length (bp)
ZnT1 / NM_022853.2 / ACACGCTAGTGGCTAACACC / TGTCCAGCCCTGTTCTCTTC / 157
ZnT2 / NM_001083122.1 / ACTGTCATGCGCAGAAGGAT / GCGAAATCAGTGAGCAGGTG / 181
NM_012890.2
ZnT3 / NM_00103243.1 / GGCCTGCGATTGAAGAGTC / AAGATGAAGCACACGACGCA / 179
ZnT4 / NM_172066.1 / ACTTGTGGCATTCACAACGC / GCATGTGCACTATGGCAGTG / 188
ZnT5 / NM_001106404.1 / TGGAGTGCTCACCAACAGTC / AAATCCGGGTGGCTTTCCAT / 122
ZnT6 / NM_001277279.1 / CAGCTTGTGCGGCATTATCC / CTGACGCAGTGTCTACAGCA / 152
ZnT7 / NM_001191715.1 / TGTAATTGCCTCCGCCATCA / TCCAACGAGGGAGGAGTTCT / 156
ZnT9 / NM_001109088.1 / GCAGCCAGGGATGGAAGAAT / GGCTTTGTCAGTTCTCGGAGT / 158
ZIP1 / NM_001134577.1 / CCACATCCGGAAGAGACGAG / CAGCCAGGCCCTCAAATACA / 175
ZIP3 / NM_001008356.1 / CGAAGTGCAGTACCCCTGAG / CCAGGAAGGTGACGAACAGG / 178
ZIP4 / NM_001077669.1 / AGCTCCATGCTACCCACAAC / GGAGCTGCAGGCTTCACTTA / 173
ZIP5 / NM_001108728.1 / GGACACCAAGAGCCTCCAACTG / TTCCTGCCCAGACATCCACACT / 103
ZIP6 / NM_001024745.1 / CGGGAGACGCACCAAAGATA / CTTCCTGGGCTCACTCACAG / 124
ZIP7 / NM_001164744.1 / CGATGCGTCTGCAACTCTTG / TAACGGGGATGCCTCTCTCA / 200
ZIP8 / NM_001011952.1 / GGGCAACAACTTTGCTCCAA / AAGATGGCAGTGAATCCGGT / 186
ZIP9 / NM_001034929.1 / GGAGCAGCAGCCTCTACTTCAC / GGAAGGAAACCAGCCCAAATGC / 100
ZIP10 / NM_001108796.2 / ACTGCCACACGAGTTAGGTG / CGTGATCGCGAAGATCCAGA / 172
ZIP11 / NM_001013042.1 / TTGGACCCTGCATTGGTGAA / CTTCCTCTGGTACACCTCGC / 120
ZIP13 / NM_001039196.1 / GTACGCAATCCCCCAAAGGA / GGAGAGGGGGATTTAGGTGC / 144
ZIP14 / NM_001107275.1 / ACTGATTAACCTGGCCTCGC / CGTTAGAGAGCAGCGTTCCA / 119

ZnT: Zinc transporter; ZIP: Zrt-, Irt-related proteins

Table 2 Real-time PCR result for zinc transporters in 3 groups

Name / Normal control (N) / CoCl2-treated (CC) / CoCl2+EPO-treated (CC+E) / p-value
Mean / SEM / n / Mean / SEM / n / Mean / SEM / n / CC Vs. N / E Vs. CC
ZnT1 / 1 / 0.09 / 5 / 4.59 / 0.21 / 5 / 6.97 / 0.58 / 5 / 2.953E-07 / 0.005
ZnT2 / 1 / 0.15 / 6 / 0.94 / 0.10 / 3 / 1.19 / 0.13 / 4 / 0.784 / 0.192
ZnT3 / 1 / 0.19 / 4 / 0.95 / 0.17 / 4 / 1.15 / 0.08 / 4 / 0.846 / 0.313
ZnT4 / 1 / 0.06 / 6 / 1.02 / 0.07 / 5 / 1.08 / 0.07 / 5 / 0.811 / 0.539
ZnT5 / 1 / 0.08 / 6 / 1.33 / 0.16 / 5 / 0.99 / 0.03 / 5 / 0.084 / 0.069
ZnT6 / 1 / 0.07 / 6 / 1.05 / 0.07 / 6 / 1.17 / 0.05 / 6 / 0.610 / 0.170
ZnT7 / 1 / 0.06 / 6 / 1.14 / 0.06 / 6 / 1.16 / 0.05 / 6 / 0.123 / 0.836
ZnT9 / 1 / 0.08 / 6 / 1.00 / 0.08 / 6 / 1.19 / 0.04 / 5 / 0.995 / 0.062
ZIP1 / 1 / 0.13 / 4 / 1.64 / 0.15 / 5 / 1.51 / 0.24 / 3 / 0.017 / 0.637
ZIP3 / 1 / 0.14 / 6 / 0.63 / 0.05 / 6 / 0.79 / 0.08 / 6 / 0.033 / 0.139
ZIP4 / 1 / 0.45 / 3 / 1.28 / 0.31 / 6 / 1.08 / 0.15 / 5 / 0.625 / 0.599
ZIP5 / 1 / 0.29 / 5 / 0.63 / 0.13 / 6 / 0.62 / 0.10 / 5 / 0.251 / 0.958
ZIP6 / 1 / 0.18 / 5 / 0.92 / 0.13 / 5 / 1.16 / 0.08 / 5 / 0.724 / 0.147
ZIP7 / 1 / 0.15 / 5 / 1.25 / 0.19 / 5 / 1.72 / 0.22 / 5 / 0.338 / 0.143
ZIP8 / 1 / 0.05 / 5 / 1.05 / 0.15 / 4 / 0.51 / 0.09 / 5 / 0.731 / 0.015
ZIP9 / 1 / 0.11 / 4 / 1.26 / 0.10 / 6 / 1.21 / 0.12 / 5 / 0.127 / 0.751
ZIP10 / 1 / 0.22 / 4 / 0.37 / 0.06 / 6 / 0.39 / 0.04 / 6 / 0.010 / 0.818
ZIP11 / 1 / 0.15 / 4 / 1.23 / 0.21 / 5 / 1.19 / 0.12 / 5 / 0.428 / 0.896
ZIP13 / 1 / 0.32 / 4 / 1.28 / 0.27 / 4 / 1.25 / 0.11 / 6 / 0.533 / 0.905
ZIP14 / 1 / 0.17 / 5 / 0.79 / 0.10 / 6 / 0.59 / 0.07 / 6 / 0.297 / 0.135

SEM: standard error of mean

Fig. 1 Cell viability assay of rMC-1 cells treated with ZnCl2 or ZnCl2+EPO. The rMC-1 cells were treated with ZnCl2 (125 μM) or ZnCl2 (125 μM) + EPO (40 U/mL) for 24 hours, and then the cell viability was examined with ATP content measurement using the CellTiter-Glo® Assay. The cell viability was expressed as the percentage of normal control (N, without treatment), which was defined as 100%. Data were expressed as mean ± SE (n=6). * means P < 0.05 compared with ZnCl2-treated rMC-1 cells.

Fig. 2 DNA-binding activity and immunostaining of HIF-1α in rMC-1 cells. (A) The sequence of HIF-1α binding elements (wide type). The mutated sequence was underlined and shown in italic. (B) EMSA competition experiments. Lane 1: the mobility of the labeled wild-type probe without nuclear extracts (negative control); lane 2: the mobility of the nuclear extract of CoCl2-treated rMC-1 cells with a 100-fold molar excess of unlabeled wide-type probe (cold probe), competed successfully with the wild-type probe; lane 3: the mobility of the nuclear extract of CoCl2-treated rMC-1 cells with a 100-fold molar excess of mutated probe, which does not bind to HIF-1α (mutant probe); lane 4: the nuclear extract of normal control with labeled wild-type probe; lane 5: the nuclear extract of CoCl2-treated rMC-1 cells with labeled wild-type probe; lane 6: the nuclear extract of EPO-treated rMC-1 cells with labeled wild-type probe. (C) The immunostaining of HIF-1α (red) in three groups. The nucleus was counterstained with DAPI (blue). Bar is 10 μm. N: Normal control; CC: CoCl2 (200 μM)-treated rMC-1 cells; CC+E: CoCl2+EPO (40 U/mL) treatment.