SUPPLEMENTARY INFORMATION (FIG S1, S2, S3, S4).
SUMMARY:
Fig S1 shows the effects of hypoxia and DFO on mRNA levels of ISCU and COX10 in
four cell lines.
Fig S2 confirms that ISCU and COX10 are potential targets of miR-210 in three
additional cell lines, HCT-/-, HT29 and MCF-7.
Fig S3 demonstrates that miR-210 does not regulate mRNA levels of other components
of the electron transport chain, NDUFA4, SDHALP2 and COX15 in HCT116 cells.
Fig S4 demonstrates that gene knockdown using siRNA of ISCU or COX10 caused
downregulation of mitochondrial respiration activity, but ROS level was not significantly
altered. Meanwhile, forced expression of coding region of ISCU1/2 can partially reverse
the effect of miR-210 on mitochondria respiration.
FIGURES LEGENDS
Fig S1: Hypoxia or DFO treatment can decrease ISCU and COX10 mRNA levels. Four
cell lines were treated under hypoxia for 24 hours or DFO for 12 hours. ISCU (A) and
COX10 (B) mRNA levels were analyzed by q-PCR. Data shown are the means and
standard deviations of triplicate qPCR reactions. Corresponding p values are shown.
DFO, deferoxamine mesylate.
Fig S2: ISCU and COX10 are potential targets of miR-210. (A) HCT-/-, HT29 and
MCF-7 cells were treated with miR-210 mimic under normoxic condition or mir-210
hairpin inhibitor under hypoxic condition. After 24 hours, gene expression level of ISCU
(A) and COX10 (B) were analyzed by qPCR. MiRNA mimic negative control or hairpin
inhibitor negative control was applied as control. Data shown are the means and standard
deviations of triplicate qPCR reactions. Corresponding p values are shown.
Fig S3: NDUFA4, SDHALP2 and COX15 mRNA levels are not regulated by miR-210.
HCT116 cells were treated with miR-210 mimic under normoxic condition or miR-210
hairpin inhibitor under hypoxic condition. After 24 hours, expression levels of NDUFA4
(A), SDHALP2 (B) and COX15 (C) were analyzed by qPCR. MiRNA mimic negative
control or hairpin inhibitor negative control was applied as control. Data shown are the
means and standard deviations of triplicate qPCR reactions. Corresponding p values are
shown.
Fig S4: Knockdown of ISCU and COX10 alters mitochondrial respiration activity. (A)
Oxygen consumption assays were performed on HCT116 cells treated with siRNA for
ISCU and COX10 for 48 hours. (B) H2O2 levels of HCT116 cells treated with siRNA for
ISCU and COX10 for 24 hours were measured by flow cytometry using fluorescent dye
DCF-DA. (C) Stable HCT116 cells expressing ISCU1/2 or empty vector were transfected
with miR-210 mimic and negative control for 48 hours. Western blot analysis was
performed to monitor ISCU protein levels. (D) Stable HCT116 cells expressing ISCU1/2
or empty vector were transfected with miR-210 mimic and negative control. Oxygen
consumption assays were performed after 48 hours. Data shown are the means and
standard deviations of duplicate experiments. Corresponding p values are shown. siRNA,
small interfering RNA. DCF-DA, CM-H2DCFDA.