EMBO/soda-nel huggingface dataset:
Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone (2019)
Figure 1

Figure 1 - CCN1 is specifically expressed in ependymal cells in the adult V-SVZ.

A Immunostaining for CCN1 (green) in the adult brain coronal section. Scale bar: 50 μm. The insert refers to the same image with DAPI staining to show cell nuclei (blue). LV, lateral ventricle.

B Adult V-SVZ whole-mounts stained for CCN1 (white) and S100β (green). Scale bar: 10 μm.

C Adult V-SVZ whole-mounts stained for CCN1 (white), GFAP (green) and VCAM1 (magenta). Arrowhead points to a VCAM1+ B1 cell surrounded by CCN1-expressing ependymal cells. Scale bar: 10 μm.

D Confocal images tracing a VCAM1+GFAP+ cell reveal CCN1 expression is absent in B1 cells. Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit. White arrowheads point to the process of the B1 cell. Scale bar: 10 μm.
Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone (2019)
Figure 2

Figure 2 - CCN1 restricts NSC pool size and niche capacity in the adult V-SVZ.

A V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm.

B V-SVZ whole-mounts stained for γ-tubulin (cyan) and GFAP (red). Arrowheads point to B1 cells in the pinwheel center. Scale bars: 500 μm (left), 50 μm (middle), 10 μm (right).

C Densities (cells/mm2) of B1 cells in the adult V-SVZ. n=4, ***p=0.0005.

D V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).

E Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ. n=4, **p=0.002.

F Densities (units/mm2) of NSC units in the adult V-SVZ. n=4, **p=0.0022.

G Distribution of NSC units with different numbers of B1 cells. n=4, ****p<0.0001 (1 B1 cell/unit), **p=0.0025 (2 B1 cells/unit), **p=0.0063 (3 B1 cells/unit), *p=0.0385 (4 B1 cells/unit).

Data information: In (C, E, F and G), data are presented as mean ± SEM. Unpaired Student's t-test.
Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone (2019)
Figure 3

Figure 3 - The enlarged V-SVZ niche persists till aging in Ccn1cKO mice

A Densities (cells/mm2) of B1 cells in the aged V-SVZ. n=3, **p=0.0099.

B Densities (cells/mm2) of VCAM1+ B1 cells in the aged V-SVZ. n=3, **p=0.0041.

C V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white) in aged mice. Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).

D Densities (units/mm2) of NSC units in the aged V-SVZ. n=3, **p=0.0093.

E Average numbers of B1 cells in a NSC unit. n=3, n.s. not significant.

Data information: In (A, B, D and E), data are presented as mean ± SEM. Unpaired Student's t-test.
Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone (2019)
Figure 4

Figure 4 - Loss of CCN1 transiently enhanced NSC proliferation and reduced differentiation.

A Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 2 days after TAM treatment. n=4, *p=0.0141. V-SVZ whole-mounts stained for GFAP (red) and Ki67 (cyan). Arrowheads point to dividing GFAP+ cells. Scale bar: 10 μm.

B Numbers of ASCL1+ cells in V-SVZ coronal sections 2 days after TAM treatment. n=3, **p= 0.0087. V-SVZ coronal sections stained for ASCL1 (red) and nuclei (blue). Scale bar: 50 μm.

C Densities (cells/mm2) of BrdU+ cells in the OB granule cell layer (GCL) 2 days after TAM treatment. n=4 (Ctrl) and 3 (Ccn1cKO), **p=0.0086. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.

D Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 4 weeks after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.

E Numbers of ASCL1+ cells in V-SVZ coronal sections 4 weeks after TAM treatment. n=3, n.s. not significant. Scale bar: 20 μm.

F Densities (cells/mm2) of BrdU+ cells in the OB GCL 4 weeks after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.

G Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 16 months after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.

H Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.

Data information: In (A-H), data are presented as mean ± SEM. Unpaired Student's t-test.
Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone (2019)
Figure 5

Figure 5 - Loss of CCN1 increased quiescent NSCs in the V-SVZ

A Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm.

B Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm.

C Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm.

D Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.

E Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm.

F Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm.

G Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.

Data information: In (A-G), data are presented as mean ± SEM. Unpaired Student's t-test.
Ependyma-expressed CCN1 restricts the size of the neural stem cell pool in the adult ventricular-subventricular zone (2019)
Figure 6

Figure 6 - CCN1 regulates NSC pool expansion through EGFR signaling.

A Representative FACS plots showing gating strategy for the analysis of cell types in the adult V-SVZ.

B Total number of V-SVZ cells isolated from one mouse. n=3, n.s. not significant.

C Representative FACS plots showing aNSCs (GFP+EGFR+), qNSCs (GFP+EGFR-) and C cells (GFP-EGFR+) in the adult V-SVZ.

D Percentage of each cell population in the adult V-SVZ. n=3, **p=0.0014 (GFP+EGFR-), ***p=0.0002 (GFP+EGFR+), *p=0.0111 (GFP-EGFR+), ***p=0.0006 (GFP+), n.s. not significant.

E Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001.

F Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002.

G V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Scale bar: 50 μm.

Data information: In (B, D, E and F), data are presented as mean ± SEM. Unpaired Student's t-test.

Expanded View Figure Legends
In vivo single cell transcriptomics reveal K. pneumoniae skews lung macrophages to promote infection (2022)
Figure 2

Figure 2. K. pneumoniae-induced M(Kp) polarization is STAT6 dependent.

A. Immunoblot analysis of the levels of the indicated proteins in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.

B. klf4 mRNA levels were assessed by qPCR in wild-type iBMDMs infected with Kp52145 for 1, 3 or 5 h

C. Immunoblot analysis of Klf4 and tubulin levels in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.

D-F. Analysis by flow cytometry of the levels of M(Kp) markers expressed by IMs from PBS mock infected mice (black dots), and by IMs from infected mice (blue dots) with and without associated Kp52145. D. Percentage of positive cells for Arg1. E. Percentage of positive cells for Fizz1. F. Percentage of positive cells for iNOS.

G. arg1 mRNA levels were assessed by qPCR in wild-type and stat6-/- iBMDMs non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

H. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type and stat6-/- iBMDMs for 60 or 120 min.

I. il10 mRNA levels were assessed by qPCR in wild-type (WT) and stat6-/- iBMDMs non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

Data information: For all infections, after 1 h contact, medium replaced with medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. qPCR and flow cytometry values are presented as the mean ± SEM of three independent experiments measured in duplicate. Images are representative of three independent experiments. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
In vivo single cell transcriptomics reveal K. pneumoniae skews lung macrophages to promote infection (2022)
Figure 3

Figure 3. STAT6 promotes K. pneumoniae infection.

A. Kp52145 adhesion to wild-type (WT) and stat6-/- iBMDMs. Cells were infected with Kp52145 for 30 min, washed, cell lysed with saponin, and bacteria quantified after serial dilution followed by plating on LB agar plates.

B. Phagocytosis of Kp52145 by wild-type (WT) and stat6-/- iBMDMs. Cells were infected for 30 min, wells were washed, and it was added medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. After 30 min, cells were washed, cell lysed with saponin, and bacteria quantified after serial dilution followed by plating on LB agar plates.

C. Kp52145 intracellular survival in wild-type (W)T and stat6-/- 5 h after addition of gentamycin (30 min of contact). Results are expressed as % of survival (CFUs at 5 h versus 30 min in stat6-/- cells normalized to the results obtained in wild-type macrophages set to 100%).

D. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and stat6-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments.

E. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.

F. Bacterial burden in the lungs of Kp52145infected wild-type and stat6-/- mice (n=7 per group) 24 h post infection. Each dot represents one animal.

G. Bacterial dissemination assessed by quantifying CFUs in the spleens of the same mice as in F.

Η. C56BL/6 mice were treated 24 h prior to infection with the STAT6 inhibitor AS1517499 (10mg/kg in 200 μL volume by i.p) and 6 h post infection (5 mg/kg in 30 μl volume intranasally) or vehicle control DMSO (n=8 per group). Bacterial burden was established by serial dilutions of lung homogenates on Salmonella-Shigella agar. Each dot represents one animal.

I. Bacterial dissemination assessed by quantifying CFUs in the spleens from the same infected mice treated with AS1517499 or vehicle control DMSO.

Data information: Values are presented as the mean ± SEM of three independent experiments measured in duplicate. ***P ≤ 0.001; ** P ≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons determined using unpaired t test.
In vivo single cell transcriptomics reveal K. pneumoniae skews lung macrophages to promote infection (2022)
Figure 4

Figure 4. K. pneumoniae-induced M (Kp) polarisation is dependent on TLR signalling and the TLR adaptors MyD88, TRAM and TRIF.

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min.

B. Immunoblot analysis of Arg1and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min.

C. arg1 mRNA levels were assessed by qPCR in wild-type (WT) and tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

D. fizz1 mRNA levels were assessed by qPCR in wild-type (WT) and tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

E. il10 mRNA levels were assessed by qPCR in wild-type (WT) and tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

F. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min.

G. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min.

H. arg1 mRNA levels were assessed by qPCR in wild-type (WT), myd88-/-, tram/trif-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

I. fizz1 mRNA levels were assessed by qPCR in wild-type (WT), myd88-/-, tram/trif-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

J. il10 mRNA levels were assessed by qPCR in wild-type (WT), myd88-/-, tram/trif-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

Data information: For all infections, after 1 h contact, medium replaced with medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. Error bars are presented as the mean ± SEM of three independent experiments in duplicate. Images are representative of three independent experiments. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
In vivo single cell transcriptomics reveal K. pneumoniae skews lung macrophages to promote infection (2022)
Figure 5

Figure 5. K. pneumoniae exploits type I IFN signalling to induce M(Kp) polarisation.

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min.

B. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min.

C. arg1 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

D. nos2 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

E. pparg mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

F. fizz1 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

G. il10 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1-/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.

H. Percentage of wild-type (WT) and ifnar1-/- iBMDMs with and without associated Kp52145 positive for Arg1 1, 3 or 5 h post infection. Kp52145 was tagged with mCherry.

I. Percentage of wild-type (WT) and ifnar1-/- iBMDMs with and without associated Kp52145 positive for CD206 1, 3 or 5 h post infection. Kp52145 was tagged with mCherry.

J. Percentage of wild-type (WT) and ifnar1-/- iBMDMs with and without associated Kp52145 positive for MHCII 1, 3 or 5 h post infection. Kp52145 was tagged with mCherry.

K. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf3-/- for 60 or 120 min.

L Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf31-/- for 60 or 120 min.

Data information: For all infections, after 1 h contact, medium replaced with medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. Error bars are presented as the mean ± SEM of three independent experiments in duplicate. Images are representative of three independent experiments. ****P ≤ 0.0001; ***P ≤ 0.001; **P≤ 0.01; *P ≤ 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.
In vivo single cell transcriptomics reveal K. pneumoniae skews lung macrophages to promote infection (2022)
Figure 7

Figure 7. K. pneumoniae-governed M(Kp) is dependent on the capsule polysaccharide.

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the LPS O-polysaccharide mutant, strain 52145-Δglf, for 60 or 120 min.

B. Percentage of wild-type macrophages with and without associated Kp52145 or 52145-∆glf positive for CD206 5 h post infection. Bacteria were tagged with mCherry.

C. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.

D. arg1 mRNA levels were assessed by qPCR in wild-type macrophages non-infected (ni) or infected Kp52145, the CPS mutant, strain 52145-ΔwcaK2, the complemented strain, 52145-ΔwcaK2/pGEMTman (Comp) for 1, 3 or 5 h.

E. Percentage of wild-type macrophages with and without associated Kp52145, 52145-ΔwcaK2, or the complemented strain positive for Arg1 5 h post infection. Bacteria were tagged with mCherry.

F. Percentage of wild-type macrophages with and without associated Kp52145, 52145-ΔwcaK2, or the complemented strain positive for CD206 5 h post infection. Bacteria were tagged with mCherry.

G. Percentage of wild-type macrophages with and without associated Kp52145, 52145-ΔwcaK2, or the complemented strain positive for MHCII 5 h post infection. Bacteria were tagged with mCherry.

Data information: For all infections, after 1 h contact, medium replaced with medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. Error bars are presented as the mean ± SEM of three independent experiments in duplicate or triplicate. Images are representative of three independent experiments. Statistical analysis were carried out using one-way ANOVA with Bonferroni contrast for multiple comparisons test. ****P ≤ 0.0001; ***P ≤ 0.001; **P ≤ 0.01*P ≤ 0.05; ns, P > 0.05 for the indicated comparisons.
Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis (2015)
Figure 1

Figure 1 - Bax forms ring-like structures on fragmented mitochondria during apoptosis.

A Left: Overlay image of a confocal recording of an apoptotic U2OS cell surrounded by non-apoptotic cells labeled with Bax (green) and Tom22 (red). Bax translocates to the mitochondria in apoptotic cells. Right: Individual display of the two color channels as shown in the overlay image. The asterisk denotes the apoptotic cell. Scale bars: 10 µm.

B, C Dual color image of an apoptotic U2OS cell decorated with antibodies against Bax (green) and Tom20 (red). The Bax signal was recorded in the STED mode and the Tom20 signal in the diffraction-limited confocal mode. (B) and (C) display the same data set. The small images are magnifications of the areas indicated by the rectangles. In (B) the color table was adjusted to the brightest Bax signal in the image resulting in a clear display of the clusters. In (C) the color table was adjusted such that the dimmer Bax-rings are visible and the clusters are saturated. The arrows point to Bax-rings. Scale bars: 1 µm.

D Optical sections of an apoptotic mitochondrion labelled for Bax (green) and Tom22 (red). Shown are three optical sections and a maximum intensity projection of the entire image stack. Scale bar: 0.5µm.

E Bax-rings on apoptotic mitochondria of various mammalian cell types. Shown are apoptotic mitochondria labelled for Bax (green) and Tom22 (red) in HT1080, SH-SY5Y, HeLa and CV-1 cells, as indicated. Scale bars: 0.5 µm.

F Quantification of the diameter of Bax-rings in apoptotic U2OS cells. Only the long ring-axis was measured. Bax-rings smaller than 200 nm exist, but were not considered.
Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis (2015)
Figure 2

Figure 2 - The area enclosed by a Bax-ring is devoid of the mitochondrial outer membrane protein Tom22.

Apoptotic mitochondria were decorated with antibodies against Bax (green) and Tom22 (red). The Bax signal was recorded in the STED mode. Shown are the individual color channels as well as an overlay (from top to bottom) of several apoptotic mitochondria. We used Lagrange interpolation to double the number of pixels. Note that throughout this manuscript except for contrast stretching no image manipulation was applied. Scale bar: 0.5 µm.
Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis (2015)
Figure 3

Figure 3 - The formation of Bax-rings is strictly correlated to cytochrome c release in apoptotic wild type cells.

A, B Three-color images of mitochondria of unchallenged (A) and apoptotic (B) U2OS cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows denote Bax-rings. Scale bars: 2 µm.
Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis (2015)
Figure 4

Figure 4 - In apoptotic Drp1 knockdown cells, the formation of Bax-rings is not sufficient for full cytochrome c release.

A In apoptotic Drp1 knockdown U2OS cells, cytochrome c release is delayed. Confocal overview image of an apoptotic Drp1 knockdown cell labelled with antibodies against Tom20 (red), cytochrome c (blue) and Bax (green). Large image: Overlay of all three channels. Small images: Magnifications of the area indicated by the rectangle in the large image. Shown are the individual channels, as indicated. The arrows point to mitochondria that had not released cytochrome c; the asterisks denote mitochondria that had released cytochrome c. Scale bars: 10 µm (overview) and 2 µm (magnifications).

B In Drp1 knockdown cells Bax-ring formation and cytochrome c release is not correlated. Three-color images of apoptotic mitochondria of Drp1 knockdown cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows point to Bax-rings. Scale bar: 1 µm.
Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis (2015)
Figure 5

Figure 5 - The redistribution of the MICOS core components Mic27 and Mic60 precedes the release of cytochrome c in apoptotic Drp1 knockdown cells.

Drp1 knockdown cells were treated with DMSO (control) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax, cytochrome c, and Mic27 or Mic60 (see Appendix Figure S4). Three-color images were taken, whereby Mic27 or Mic60 were recorded in the STED mode. Relying on the Bax and the cytochrome c signals, we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release. The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity, which is a sensitive measure for the distribution of the labelled protein. The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean.