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. 2023 Oct 11;24(12):e57228. doi: 10.15252/embr.202357228

Figure 2. MTIF3 potentiates tetracycline‐induced cell survival in ND1 mutant cells.

Figure 2

  • A
    CRISPR‐Cas9 KO of MTIF3 in ND1 cybrid cells reduces doxycycline's ability to promote survival (error bars represent the average ± s.e.m. of n = 3 biological replicates). Statistical significance (P < 0.05) between doxycycline treated and untreated cells across NTC and sgMTIF3 (sg1‐sg3) was calculated using two‐way ANOVA, multiple comparisons.
  • B
    CRISPR‐Cas9 KO of MALSU1 and MTIF3 drastically reduce the ability of tetracyclines to promote survival when compared to LOR8F8‐KO (error bars represent the average ± s.e.m. of n = 6 biological replicates). Statistical significance (P < 0.05) between doxycycline treated and untreated cells across NTC, sgMALSU1, sgLOR8F8, and sgMTIF3 cells was calculated using two‐way ANOVA, multiple comparisons.
  • C
    MALSU1 is recruited to the mitochondria at early time points. Crude mitochondria (mitochondria/ER) were isolated from Expi293F cells at varying time points following doxycycline treatment. Note maximum mitochondrial MALSU1 at the 12‐h time point.
  • D
    Mitoribosome isolation in Expi293F cells indicates doxycycline promotes the accumulation of MALSU1 staring at 1 h, with drastic increases at the 12‐h time point. Note mitochondrial accumulation of MALSU1 at 12 h. MTIF3 expression, along with mitochondrial and mitoribosome accumulation were unchanged with doxycycline.
  • E, F
    (E) Mitochondria were isolated from ND1 mutant cybrid cells exposed to galactose with or without doxycycline for varying time points, with rapid mitochondrial accumulation of MALSU1 after 1 h, that maximizes from 3 to 24 h of doxycycline treatment. Note subtle increases in MTIF3, along with MRPL4 and MRPL6 at the 3‐h time points. (F) Matched glucose controls at the 3‐ and 24‐h time point illustrate a decrease in mitochondrial MALSU1, MRPL4, and MRPS16 upon galactose treatment that is stabilized with doxycycline. Note increased whole cell expression level of MALSU1 in doxycycline treated cultures at the 24‐h time point.

Source data are available online for this figure.