EMBO/soda-nel huggingface dataset:
Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission (2022)
Figure 1

Figure 1. The abundance of somatic and synaptic PI(3)P-positive endosomes is regulated by neuronal activity via Cdk5

A Representative 3-color STED images of synapses of cultured hippocampal neurons fixed and stained for Synapsin1/2 (presynaptic marker), PI(3)P and Homer (postsynaptic marker). Scale bar, 0.5 µm. PI(3)P-positive structures appear in both pre- and postsynaptic compartments.

B Line scan fluorescence intensity profiles of single synapses from the images in A, normalized to maximum value for each channel.

C Representative 3-color confocal images of cultured hippocampal neurons fixed and stained for VGlut1, VGAT and PI(3)P. Arrows indicate PI(3)P puncta in VGlut1+ or VGAT+ presynaptic terminals. Scale bar, 10 µm.

D PI(3)P intensity in VGlut1+ and VGAT+ presynaptic terminals. PI(3)P is equally abundant in excitatory and inhibitory presynaptic terminals. N = 3 independent experiments (≥ 15 images per condition); Student's t-test.

E Relative PI(3)P levels in the MAP2+ (Soma) and Syp+ (Synapse) areas in DMSO and VPS34IN1-treated neurons. N = 3 independent experiments (≥ 35 images per condition); Student's t-test.

F Cultured hippocampal neurons were field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for MAP2, Synaptophysin (Syp), and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm. PI(3)P puncta that do not overlap with neuronal soma and synapses belong to astrocytes in culture and are disregarded from the quantification.

G Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in F. N = 5 independent experiments; (≥ 40 images per condition); Student's t-test.

H Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons treated with DMSO or Gabazine (10 µM) for 24 h. N = 3 independent experiments (≥ 65 images per condition); Student's t-test.

I Cultured hippocampal neurons treated with AP5 (20 µM), CNQX (10 µM) or TTX (0.2 µM) for 24 h, fixed and stained for MAP2, Syp and PI(3)P. Arrows indicate PI(3)P puncta in synapses. Scale bar, 10 µm.

J Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) from the images in I. N ≥ 4 independent experiments (≥ 60 images per condition); Student's t-test.

K Relative intensity of PI(3)P in neuronal somata (MAP2+) or synapses (Syp+) of cultured hippocampal neurons were treated with Roscovitine (10 µM) for 1 h and field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-). N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Fisher's Least Significant Difference Test.

Data information: Data presented as mean ± SEM in all panels; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p, t, q, df values are provided in the Source Data Statistics file.
Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission (2022)
Figure 2

Figure 2. | Loss of PI(3)P leads to reduced basal synaptic transmission and perturbs SV endocytosis

A Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%) or the VPS34 inhibitors, VPS34IN1 (3 µM) or SAR405 (20 µM). Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. VPS34 inhibition causes reduced excitatory synaptic transmission down to 46.0 ± 4.1 % and 55.0 ± 4.4 % for VPS34IN1 and SAR405 at the end of 60-min treatment. N = 6 slices per condition from ≥ 4 mice; Student's t-test.

B Increased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses after VPS34 inhibition. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below, indicating an increased facilitation of the second response in VPS34IN1-treated acute hippocampal slices. PI(3)P depletion causes a significant facilitation of the second fEPSP, indicative of reduced initial release probability. N = 6 slices per condition from 6 mice; Two-way ANOVA.

C Synaptophysin-pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs (20 Hz, 1 s) with 30 s gap between each stimulus. Neurons are acutely treated with DMSO (0.1 %) or VPS34 inhibitor1 (50 µM) starting from the onset of 2nd stimulation. The timing of the treatment and the stimulation trains are indicated.

D Quantification of data from C. Net difference of Synaptophysin-pHluorin fluorescence 2 s before and 28 s after each stimulation (2 s before the next stimulation) to estimate relative surface retention of SVs. SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1, leading to progressive accumulation of Synaptophysin-pHluorin signal. N = 4 or 5 independent measurements per condition; Student's t-test.

Data information: Data presented as mean ± SEM in all panels; *p < 0.05; **p < 0.01; ***p < 0.001; p, t, q, df values are provided in the Source Data Statistics file.
Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission (2022)
Figure 4

Figure 4. Depletion of PI(3)P impairs SV endocytosis in stimulated synapses

A-D Normalized Synaptophysin-pHluorin responses from neurons treated with VPS34IN1 (10 µM for 1h prior to imaging) and stimulated with four consecutive trains of 50 APs (10 Hz, 5 s) in A or 200 APs (40 Hz, 5 s) in C. PI(3)P depletion in VPS34IN1-treated neurons causes the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D. Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive stimulation trains. N ≥ 4 independent experiments; Two-way ANOVA; Sidak's Multiple Comparison Test.

E Cultured hippocampal neurons pre-treated with DMSO (0.1%) or VPS34IN1 (10 µM, 1 h) were either field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for surface-stranded Synaptotagmin-1, total Synaptotagmin-1, and vGlut1. Scale bar, 10 µm.

F Fraction of endogenous Synaptotagmin-1 stranded on the surface of presynaptic nerve terminals from the images shown in E. N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test.

G Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405 (20 µM). Data are similar to those shown in C and D. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test.

H Exogenous supply of membrane-permeant PI(3)P acutely rescues defective Synaptophysin-pHluorin endocytosis induced by pharmacological inhibition of VPS34-mediated PI(3)P synthesis in hippocampal neurons. Data are similar to those shown in C and D; where indicated exogenous PI(3)P-AM (20 µM) was supplied in parallel to VPS34IN1 (10 µM) for 1 h prior to imaging. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test.

I Kinetic block of Synaptophysin-pHluorin endocytosis at hippocampal synapses depleted of VPS34. Hippocampal neurons depleted of endogenous VPS34 by specific shRNA (shVPS34) or treated with non-targeting control shRNA (shCtrl) and stimulated as in C and analyzed as in D. N =3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test.

Data information: Data presented as mean ± SEM in all panels; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p, t, q, df values are provided in the Source Data Statistics file.
Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission (2022)
Figure 5

Figure 5. Impaired SV endocytosis upon PI(3)P depletion is activity- and Calpain 2-dependent

A-C The kinetic block of SV endocytosis at excitatory synapses depleted of PI(3)P depends on prior neuronal activity and Ca2+. VPS34IN1-induced defects in SV endocytosis are reverted by parallel treatment with TTX (0.2 µM) in A and EGTA (2 mM) in B and AP5 (20 µM) in C, in Synaptophysin-pHluorin-expressing neurons stimulated and analyzed as in Fig 4C and D. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test.

D Overexpression of dominant-negative Rab5 (Rab5DN), but not constitutively active Rab5 (Rab5CA), rescues the progressive accumulation of Synaptophysin-pHluorin on the surface of VPS34IN1-treated hippocampal neurons stimulated and analyzed as in A-C. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test.

E Levels of various synaptic proteins in DMSO (0.1%) and VPS34IN1 (10 µM) treated cultured cerebellar granule neurons analyzed by western blot. VPS34 inhibition results in p35 processing to hyperactive p25, a reaction blocked by Calpain inhibitor ALLN (100 µM).

F-H Calpain inhibition by ALLN (100 µM) in F or Calpain 2 knockdown via lentiviral shRNA (shCAPN2) in H rescue the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1. Calpain 1 knockdown via lentiviral shRNA (shCAPN1) in G had no impact on VPS34IN1-induced kinetic delay of SV endocytosis. Data are similar to those shown in A-C. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test.

Data information: Data presented as mean ± SEM in all panels; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p, t, q, df values are provided in the Source Data Statistics file.
Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission (2022)
Figure 6

Figure 6. Reduced neurotransmission and block of SV endocytosis due to PI(3)P depletion is Cdk5-dependent

A Hypothetical signaling pathway for the induction of hyperactive Cdk5 induced by repression of VPS34 downstream of neuronal activity.

B Cdk5 inhibition by Roscovitine (Ros, 10 µM) rescues the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1 (10 µM). Neurons are stimulated and analyzed as in Figs 4C and D. N = 4 independent experiments (20 images per condition); Two-way ANOVA; Tukey's Multiple Comparisons Test.

C Calcineurin inhibition by Cyclosporin A (10 µM) phenocopies VPS34IN1-induced loss of PI(3)P with respect to the accumulation of Synaptophysin-pHluorin on the neuronal surface after four successive stimulation trains (40 Hz, 5 s). Data were analyzed as in B. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test.

D Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%), Roscovitine (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic transmission, akin to Roscovitine alone, and in contrast to VPS34IN1 alone (compare Fig 2A). N = 6 slices per condition from 6 mice; Student's t-test.

E Decreased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone (compare Fig 2B) . N = 6 slices per condition from 6 mice; Two-way RM ANOVA.

Data information: Data presented as mean ± SEM in all panels; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p, t, q, df values are provided in the Source Data Statistics file.
Autophagy proteins control goblet cell function by potentiating reactive oxygen species production (2013)
Figure 1

Atg5‐deficient goblet cells accumulate mucin. (A) Alcian blue‐stained sections of descending colons from control and Atg5VC mice. The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside. Bars=200 μm. (B) Quantification of average mucin area/goblet cell in control and Atg5VC mice (n=5–7 mice/group from 3 independent experiments; 100 goblet cells measured/mouse). **P0.01 as determined by the Student's t‐test. This analysis was performed on upper crypt goblet cells (above the upper dashed line in A). (C) Quantification of the average number of goblet cells per crypt (n=3 mice/group; over 100 crypts measured/mouse). (D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm. (E, F) Quantification of (E) the average granule density and (F) the average number of mucin granules/goblet cell (n=3 mice/group and 20 goblet cells evaluated/mouse). ***P0.001 as determined by the Student's t‐test. (G, H) Whole‐mount images of the mucosal surface from control and Atg5VC transverse colons. Whole‐mount colonic samples were either stained with Ulex europaeus agglutinin‐1 (UEA) that labelled mucus (G) or analysed by the scanning electron microscopy (H). Yellow arrows indicate the crypt opening orifices. Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice (insets in H show higher power image of the latter). Bars=100 μm.
Autophagy proteins control goblet cell function by potentiating reactive oxygen species production (2013)
Figure 2

Atg5‐deficient colonic epithelial spheroids accumulate cytoplasmic mucin within goblet cells. (A) Schematic for the generation of differentiated goblet cells within colonic epithelial spheroid cultures. (B) Representative whole‐mount images of single spheroids cultured in either 50% conditioned media (CM) or 5% CM+5 μM DAPT and stained for Muc2 (green) to visualize goblet cells. Bars=20 μm. (C, D) Graph of relative expression (as measured by qRT‐PCR) of Atoh1 (C) and Muc2 (D) mRNAs comparing spheroids grown in either 5% CM or 5% CM+ DAPT to spheroids grown in 50% CM (n=4–5 samples/group). Error bars indicate s.e.m. *P0.05; **P0.01 as determined by a two‐tailed Student's t‐test. (E, F) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5% CM+DAPT and stained for Muc2 (green: E) or with the lectin, UEA (red: F). Bars=20 μm. (G) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 as determined by a two‐tailed Student's t‐test). (H) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice as measured by qRT–PCR (n=4–5 samples/group). No statistically significant differences were found in Muc2 expression as determined by the Student's t‐test. (I, K) Images of FIP200f/f (I) and Atg14f/f (K) colonic epithelial spheroids treated with recombinant Tat‐Cre and stained with UEA (red). (J, L) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 and ***P0.001 as determined by a two‐tailed Student's t‐test).
Autophagy proteins control goblet cell function by potentiating reactive oxygen species production (2013)
Figure 3

LC3β is localized to EEA1‐positive multi‐vesicular vacuoles within colonic goblet cells. (A) Images of colonic sections stained for LC3β (green) from control and Atg5VC mice. The white‐dashed lines indicate the border between the basal surface of the crypt epithelium and the mesenchyme. White asterisks denote non‐specific plasma cell staining from the mouse monoclonal anti‐LC3β antibody. Bars=50 μm. (B) Immunogold transmission electron microscopic image for LC3β in a colonic goblet cell from a wild‐type mouse. The inset is a low power image of the entire goblet cell. The black box within the inset indicates the area shown in this panel. Note this area is not associated with mucin granules. Bar=500 nm. (C) Enriched KEGG pathways for Atg5‐deficient epithelial cells obtained from a microarray comparing control and Atg5VC colonic crypt base epithelium. P‐values were determined by modified Fisher's exact test. (D–G) Double label immunogold transmission electron microscopic images from wild‐type mice for LC3β (12 nm particles) and EEA1 (D), Rab7 (E), Rab11 (F), or Lamp1 (G) (18 nm particles). Bars=200 nm.
Autophagy proteins control goblet cell function by potentiating reactive oxygen species production (2013)
Figure 4

Inhibition of endocytosis results in goblet cell mucin accumulation. (A) Schematic for the generation of differentiated colonic epithelial cells grown in a monolayer. (B) Colonic epithelial monolayers from wild‐type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β (green) and EEA1 (red). Bars=5 μm. (C, D) Quantification of average number of LC3β dots per cell (C) and the percentage of LC3β dots that co‐localized with EEA1 per cell (D) (n=3 experiments/group; 25–30 cells/experiment). (E) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated. GAPDH is a loading control. Representative image is shown from n=3 experiments. (F) Images of wild‐type colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 100 μM Dynasore. Bars=20 μm. (G) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ***P0.001 as determined by the Student's t‐test.
Autophagy proteins control goblet cell function by potentiating reactive oxygen species production (2013)
Figure 5

NADPH oxidases are required for endosome/autophagosome co‐localization and control mucin granule accumulation. (A) Colonic epithelial monolayers from a wild‐type and p22 mut mouse stained with LC3β (green) and EEA1 (red). Bar=5 μm. (B, C) Quantification of average number of LC3β dots per cell (B) and the percentage of LC3β dots that co‐localized with EEA1 per cell (C) (n=3 experiments/group; 25–30 cells/experiment). (D) LC3 immunoblots of either wild‐type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated. Representative image is shown from n=3 experiments. (E) Double label immunogold transmission electron microscopic images for LC3β (12 nm particles) and p22phox (18 nm particles) showing a p22phox‐positive single membrane cytoplasmic vesicle (D) and a p22phox‐, LC3β‐positive vacuole (E). Bars=200 nm. (F) Colonic epithelial monolayers from a wild‐type mouse stained for LC3β (green) and p22phox (red) and quantification of percent LC3β dots co‐localized with p22phox per cell, Bar=5 μm. (G) Colonic epithelial cell spheroids from control and p22 mut mice stained with TRITC‐UEA to label goblet cell mucin. Bars=50 μm. (H) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. **P0.01; ****P0.0001 as determined by the Student's t‐test.
Autophagy proteins control goblet cell function by potentiating reactive oxygen species production (2013)
Figure 6

Goblet cell mucin accumulation correlates with diminished ROS production. (A) Colonic epithelial cell spheroids from p22phox−/−, Atg5VC and wild‐type mice. Wild‐type spheroids were treated with vehicle (control), 10 μM DPI (NADPH oxidase inhibitor) or 100 μM Dynasore. Intracellular ROS in live cells were labelled by 5 μM DCF. Bars=100 μm. (B) Quantification of fluorescent DCF normalized to control (n=3 independent experiments/group; 30 spheroids quantified/experiment). (C) Quantification of average area of cytoplasmic mucin from wild‐type mice treated with 3% NAC as compared to controls (n=6–7 mice/group). Error bars indicate s.e.m. *P0.05; **P0.01; ****P0.0001 as determined by the Student's t‐test. (D) TEM images of colonic epithelial monolayers treated with cerium chloride. The presence of electron dense precipitate is indicative of ROS production. Bar=500 nm.
Autophagy proteins control goblet cell function by potentiating reactive oxygen species production (2013)
Figure 7

Exogeneous ROS can restore mucin granule accumulation defects. (A) Quantification of fluorescent DCF normalized to controls (n=3 independent experiments/group; 10–30 spheroids quantified/culture). *P0.05 as determined by ANOVA with Tukey's multiple post‐test comparisons. (B) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (C) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). ***P0.001, ****P.0001 as determined by ANOVA with Tukey's multiple post‐test comparisons. (D) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT–PCR. No statistically significant differences were found in either group as determined by ANOVA. (E, G) Images of p22 mut or Dynasore‐treated colonic epithelial spheroids (mucin is labelled with TRITC‐UEA; red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (F, H) Quantification of average mucin area/goblet cell (n=6 sample/group; 30–60 cells were quantified/sample) corresponding to experiments performed using p22 mut (E) and Dynasore‐treated wild‐type spheroids (G). Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA‐AM (I) or PMA/ionomycin (J). (I, J) Quantification of average mucin area/goblet cell (n=5 sample/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ****P0.0001 as determined by the Student's t‐test (I) or ANOVA with Tukey's multiple post‐test comparisons (J).
Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders (2010)
Figure 1

Lysosomal fusion is impaired in LSD cells. (A) EGFR degradation was followed in MSD, MPS‐IIIA and WT MEFs by treating the cells with EGF for the indicated time to stimulate EGFR internalization. The cells were then immediately lysed and subjected to anti‐EGFR blotting. The amount of remaining EGFR was quantified by densitometry analysis (Image J) of the blot and expressed in the chart as % of the EGFR amount present at time T0 (100%). The values in the chart represent the mean±s.e.m. values of three independent experiments. (B) MSD, MPS‐IIIA and WT MEFs cells loaded with dextran (alexafluor‐594 conjugated) were labelled with anti‐LAMP1 antibody and the percentage of dextran co‐localizing with LAMP1 was evaluated. The chart displays merge values (means±s.e.m.) that represent the percentage of dextran co‐localizing with LAMP1 measured in 15 different cells of triplicate experiments. (C) The rate of lysosome fusion with autophagosomes was monitored in MSD, MPS‐IIIA and WT MEFs transfected with a tandem fluorescently tagged LC3 (Kimura et al, 2007). The rate of autophagosome maturation reflected the percentage of the LC3 ‘unfused’ (green/red fluorescence ratio) at each time (1 and 3 h) after bafilomycin removal (T0). The percentage of LC3 ‘unfused’ was displayed versus the value at T0 (assumed to be 100%). Values are represented as means±s.e.m. of triplicate experiments. *P0.05, Student's t‐test: (A): WT versus MSD and WT versus MPS‐IIIA; (B, C): WT versus MSD and WT versus MPS‐IIIA at each time point. Scale bar: 10 μm (B, C).