🧫 Experiment Protocol
Exploratoryacute kidney injuryTFAMHK2 cellsproposed
Gene silencing experiment using siRNA or shRNA to knockdown TFAM expression in HK2 cells to directly test the role of TFAM in mitochondrial function and cytokine release. This loss-of-function study aimed to determine whether TFAM deficiency alone could recapitulate the mitochondrial dysfunction and inflammatory responses observed with mtROS treatment. The experiment included assessment of mtDNA levels, mitochondrial respiratory capacity, and inflammatory cytokine production following TFAM silencing, with or without Mito-Tempo treatment.
PRIMARY OUTCOME
mtDNA levels, mitochondrial function, cytokine release
EXPECTED OUTCOMES
TFAM knockdown would impair mitochondrial function and increase cytokine release, and would prevent Mito-Tempo from rescuing these defects
SUCCESS CRITERIA
TFAM silencing recapitulates mtROS-induced mitochondrial dysfunction and abolishes Mito-Tempo rescue effects
PROTOCOL
TFAM knockdown using siRNA/shRNA in HK2 cells, followed by assessment of mtDNA copy number, mitochondrial respiratory function, and cytokine production, with and without Mito-Tempo treatment