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. 2023 May 25;42(14):e112817. doi: 10.15252/embj.2022112817

Figure EV5. Neither BNIP3L depletion nor iron supplementation alters mBCV occurrence in HeLa cells.

Figure EV5

  • A
    STED micrographs of a HeLa cell infected with B. abortus 544 GFP (magenta) for 72 h, then fixed and immunostained for TOMM20 (Abberior® STAR 635—green). The presented cell displays a rare but spectacular event of enlarged mBCVs with colonisation of the bacteria. Scale bars: 5 μm.
  • B
    Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (magenta) and transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM) for 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.
  • C
    Quantification of the number of TOMM20‐positive BCVs (mBCVs) per infected HeLa cells, from micrographs shown in (B). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ns: not significant (P = 0.6742).
  • D
    Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (magenta), treated or not (ctrl) with FeCl2 (500 μM) for 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.
  • E
    Quantification of the number of TOMM20‐positive BCVs (mBCVs) per infected HeLa cells, from micrographs shown in (D). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ns, not significant (P = 0.4724).
  • F
    Representative confocal micrographs of HeLa cells infected with B. abortus 544 WT for 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 633—magenta) and LC3 (Alexa Fluor 568—green). DNA (from the HeLa nucleus and B. abortus) was stained with Hoechst 33258 (red). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

Source data are available online for this figure.