EMBO/soda-nel huggingface dataset:
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf1

(a) Western blotting was performed to determine FoxO1 expression, p62 degradation and LC3-II accumulation in H1299, HCT116 and HeLa cells in response to serum starvation (SS) for 24 h or 0.5 mM H2O2 for 6 h in the presence or absence of E64 and pepstatin A (PEPS A). (b, c) Stable FoxO1 knockdown HCT116 cells, or cells with an empty RNAi plasmid, were incubated in a serum-free medium for up to 36 h (b) or treated with 0.5 mM H2O2 for up to 12 h (c); western blotting was performed to detect changes in FoxO1, p62, LC3-II or β-actin. (d) HCT116 cells were labelled with [3H]leucine for 48 h, washed and incubated for 24 h, before being subjected to serum starvation for 24 h or 0.5 mM H2O2 for 12 h in the presence or absence of 3-MA. At 12 or 24 h after treatment, the relative degradation of long-lived proteins was measured by counting radioactivity. Data in d are means ± s.d. (n = 3). Two asterisks, P 0.01; asterisk, P 0.05; NS, not significant, P > 0.05; Student's t-test. (e) Autophagic vesicles or autophagosomes in HCT116 cells or stable FoxO1-RNAi HCT116 cells, were observed by electron microscopy after 24 h of serum starvation. The bottom picture enlargement showing clearer autophagic vesicles indicated by an arrow. The graph shows a statistical analysis of autophagic vesicles in HCT116 cells with or without knockdown of FoxO1 in response to serum starvation. Data in e are means ± s.d. (n = 3). (f) LC3-immunopositive punctate signals were observed in HCT116 or in stable FoxO1-RNAi HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 treatment. (g) Quantification of LC3-positive punctate cells in f. Data in g are means ± s. d. (n = 3). (h) A rescue experiment using a specific mutated FoxO1 plasmid to validate the activity of FoxO1 in the induction of autophagy in HCT116 cells in the presence or absence of serum, with or without E64 and pepstatin A.
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf2

(a) A Flag-tagged FoxO1(WT), FoxO1(ΔDB), FoxO1(ΔDB3A) or an empty plasmid was individually transfected into GFP–LC3 pre-transfected H1299 cells, and the cellular localization of FoxO1 (upper panels) or GFP–LC3 punctate signals (lower panels) was observed with a confocal microscope. Scale bars, 10 μm. (b) The numbers of punctate GFP–LC3 cells are represented by the ratio of GFP–LC3-labelled punctate cells to overall survived H1299 cells (at least 200 cells were counted). Data in b are means ± s.d. (n = 3). (c) p62 degradation and LC3-II accumulation were evaluated by western blotting in H1299 cells transfected with exogenous FoxO1-expressing plasmids mentioned above. (d) H1299 cells were labelled with [3H]leucine for 48 h, then transfected with a control plasmid or various Flag–FoxO1 plasmids, in the presence or absence of 3-MA or E64 and pepstatin A. Degradation of long-lived proteins was measured at the indicated time, 24 h after transfection. Data in d are means ± s.d. (n = 3). (e) A GFP-tagged FoxO1(3A) or an empty plasmid was co-transfected with a FoxO1-promoter luciferase plasmid into H1299 cells. After 24 h, luciferase reporter activity of FoxO1 was measured. RLU, relative luciferase units. Data are means ± s.d. (n = 3). (f) A set of RNAi-resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1-RNAi HCT116 cells in the presence or absence of E64 and pepstatin A. Western blotting was performed to detect p62 degradation and LC3-II accumulation. (g) Flag-tagged FoxO1(WT)r, FoxO1(3A)r, FoxO1(3A-NLSm)r, FoxO1(NESm)r or an empty plasmid were individually transfected into GFP–LC3 pre-transfected HCT116 cells (FoxO1-RNAi treated), and the cellular localization of FoxO1 (upper panels) or GFP–LC3 punctate signals (lower panels) were observed with a confocal microscope. (h) The numbers of punctate GFP–LC3 cells are represented by the ratio of GFP–LC3-labelled punctate cells to overall survived HCT116 cells. Data in h are means ± s.d. (n = 3).
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf3

(a) Different FoxO1-expressing plasmids, namely FoxO1(WT), FoxO1(3A), FoxO1(ΔDB), FoxO1(ΔDB3A), FoxO1(NESm) and FoxO1(3A-NLSm), were transfected into H1299 cells and cell fractionation was performed to analyse cellular localization (N, nucleus; C, cytoplasm) of these FoxO1 mutants. PCAF is a loading control for nuclear protein, and actin is the loading control for cytoplasmic protein. (b) Quantitative PCR was performed to measure the transactivity of autophagy-related genes in transfected H1299 cells with different FoxO1-expressing plasmids (upper panel) or in stable FoxO1-RNAi or non-RNAi-treated HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 for 12 h (lower panel). All data are means ± s.d. (n = 3). (c) Different FoxO1-mutant-expressing plasmids were transfected into H1299 cells and a ChIP assay was performed to measure the binding activity of these mutated FoxO1 to the p27 promoter.
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf4

(a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.5 mM H2O2 for 12 h (lower panels); cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 and probed with anti-acetylated lysine (Ace-lyn). Both bottom panels display cytosolic FoxO1. IP, immunoprecipitation. (b) HCT116 cells were serum-starved for 24 h and the cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 or anti-SIRT2, followed by probing with anti-SIRT2 or anti-FoxO1, respectively (top and middle panels). FoxO1(WT) was also transfected into H1299 cells and the cytosolic protein was extracted, followed by co-immunoprecipitation with anti-FoxO1 and probing with anti-SIRT2 (bottom panels). (c) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid. At 48 h after transfection, the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 (upper panels), or for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine (lower panels). IB, immunoblot. (d) HCT116 cells were treated with AGK2 for 24 h at different doses, as indicated. The cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine.
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf5

(a, b) H1299 cells were transfected with an empty control plasmid or with the Flag–FoxO1(WT) plasmid. At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-mTOR, Beclin 1, Vps34 or Atg1 (a) and with Atg3, Atg4, Atg10, Atg7 or LC3 (b). (c) H1299 cells were co-transfected with Myc–Atg7, with Flag–FoxO1(WT) or with an empty control plasmid. At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-Flag, followed by probing with anti-Myc. HCT116 cells were serum-starved for 24 h (d) or treated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7. (e) Different Myc-tagged FoxO1 fragments and Flag-tagged Atg7 were co-transfected into H1299 cells, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag. (f) HCT116 cells were transfected with an empty control plasmid, an SIRT2(WT)-expressing plasmid or an SIRT2(168A)-expressing plasmid. At 24 h after transfection, cells were serum-starved for 24 h, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7.
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf6

(a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A. (b) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h, in the presence or absence of E64 and pepstatin A. Changes in FoxO1, p62 and LC3-II were determined by western blotting (upper panels), or the formation of LC3 punctate signals was observed (lower panels). HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed. Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel. Data are means ± s.d. (n = 3). (c) H1299 cells were transfected with Flag–FoxO1(WT), SIRT2(WT) or SIRT2(168A). At 24 h after transfection, the cell lysate was extracted and immunoblotted with anti-Flag, anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin. (d) HCT116 cells were transfected with a control plasmid, a SIRT2(WT)-expressing plasmid or a SIRT2(168A)-expressing plasmid. At 24 h after transfection, the cells were subjected to serum starvation for 24 h. The cell lysate was extracted and immunoblotted with anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin. (e) H1299 cells were transfected with Myc–Atg7, Flag–FoxO1(WT) or mutated Flag–FoxO1(KR). At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag. (f) H1299 cells were transfected with a control plasmid, Flag–FoxO1(WT), Flag–FoxO1(K262R), Flag–FoxO1(K265R), Flag–FoxO1(K274R) or Flag–FoxO1(KR). At 24 h after transfection in the presence or absence of E64 and pepstatin A, the cell lysate was extracted and immunoblotted with anti-Flag, anti-LC3, anti-p62 or anti-β-actin. (g) Left: H1299 cells were co-transfected with GFP–LC3, Flag–FoxO1(WT) or Flag–FoxO1(KR). The formation of GFP–LC3-labelled punctate signals was observed 24 h after transfection. Right: quantification of the ratio of GFP–LC3-labelled punctate cells to overall survived H1299 cells. Data in g are means ± s.d. (n = 3). Scale bars, 25 μm (b), 10 μm (g).
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf7

(a) H1299 cells were transfected with a control plasmid, Flag–FoxO1(WT), Flag–FoxO1(3A), Flag–FoxO1(ΔDB) or Flag–FoxO1(ΔDB3A), in the presence or absence of 10 mM 3-MA (upper panel) or in the presence or absence of 50 μM Z-VAD-fmk (lower panel); cells were stained with propidium iodide (PI) 48 h after transfection. PI-stained (dead) cells were counted by flow cytometry and the percentage cell loss was calculated. (b) FoxO1(WT) or Flag–FoxO1(ΔDB) was transfected into H1299 cells; 48 h later the autophagic vesicles were observed under an electron microscope. Enlarged images (right) show clearer autophagic vesicles indicated by arrows. Scale bars, 2 μm. (c) HCT116 cells with or without Atg5 RNAi were transfected with FoxO1(WT), and cell death was determined by staining with PI at 48 h after transfection. Data in c are means ± s.d. (n = 3). (d) The stable Atg5 knockdown H1299 cells was further transfected with a control plasmid, Flag–FoxO1(WT), or Flag–FoxO1(ΔDB), and the percentage cell loss was quantified by flow cytometry at 48 h after transfection. Data in d are means ± s.d. (n = 3).
Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity (2010)
figf8

(a) Stable H1299 cells with or without Atg7 knockdown were transfected with a control plasmid (group 1), Flag–FoxO1(ΔDB) (group 2) or Flag–FoxO1(ΔDB-3A) (group 3); 107 cells were then injected into 18 nude mice, with 6 mice per group. Seven weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Group 1: tumour weights were 1.22 ± 0.78 g (without Atg7 RNAi) and 1.31 ± 0.43 g (with Atg7 RNAi), P = 0.426. Group 2: tumour weights were 0.49 ± 0.18 g (without Atg7 RNAi) and 0.80 ± 0.20 g (with Atg7 RNAi), P = 0.017. Group 3: tumour weights were 1.00 ± 0.40 g (without Atg7 RNAi) and 1.10 ± 0.36 g (with Atg7 RNAi), P = 0.500. Data are means ± s.d. (n = 6). (b) Expression levels of FoxO1 and Atg7 conjugate in mice tumours were determined by western blotting. (c) HCT116 cells (107) or stable FoxO1-knockdown HCT116 cells (107) were injected into the left or right hind legs of three nude mice. Six weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Tumour weights were 0.08 ± 0.10 g (without FoxO1 RNAi) and 0.48 ± 0.06 g (with FoxO1 RNAi), P = 0.020. Data are means ± sd. n = 3. (d) Protein was extracted from one paired colon tumour (T) or adjacent normal tissue (N) from one patient, and western blotting was performed to detect changes in FoxO1 or p62. (e) Proteins were extracted from 16 paired samples for western blotting with anti-FoxO1 or with anti-p62 antibody. Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β-actin band. FoxO1 of normal tissue, 0.37; FoxO1 of tumour tissue, 0.10; P = 0.001, two-sided t-test. p62 of normal tissue, 0.32; p62 of tumour tissue, 1.00; P = 0.009, two-sided t-test. Data in e are means ± s.d. (n = 16). (f) A schematic model showing how FoxO1 is linked to the autophagic process.
Evidence for the nuclear import of histones H3.1 & H4 as monomers (2018)
Figure 1

Figure 1. Sub-cellular localisation of core H3.1 & H4 chaperoning components.

(A) Fractionation of nuclear and cytosolic compartments using a standard NP-40 lysis protocol and immunoblotting with chaperone specific antibodies.

(B) Immunofluorescence of histone chaperones in fixed cells using the same antibodies used in (A). Cells are segregated into those undergoing replication and those that are not. Scale bar represents 5 μm.

(C) Quantification of nuclear localisation shown in (B). Boxes represent the lower quartile, median and upper quartile. Whiskers represent 1.5 times the interquartile range.

(D) Real-time imaging of cells undergoing biochemical fractionation. sNASP is tagged with mCherry, whilst LamnA/C is tagged with EGFP. The left column of panels represents a maximum intensity projection. The time course on the right represents a plane in the z dimension reconstructed from 20 z-stacks penetrating 20 μm into the medium. Scale bars represent 5 μm.

(E) Quantification of nuclear leakage shown in (D). Z-stacks were flattened using a maximum intensity projection with the nuclear fluorescent signal over time plotted as a percentage of maximum (normalised). Data points represent the mean of 15 measurements with error bars representing the s. e. m..
Evidence for the nuclear import of histones H3.1 & H4 as monomers (2018)
Figure 6

Figure 6. Quantification of histone stoichiometries bound to sNASP, ASF1b and HAT1.

(A) HeLa cell lines stably expressing eGFP-sNASP, eGFP-ASF1 and eGFP-HAT1. Scale bars represent 10 μm.

(B) Histone chaperone immunoprecipitations separated by 15% SDS-PAGE and stained with Coomassie. Major bands that could be confidently identified by mass spectrometry are indicated. Due to the high percentage of acrylamide used, many of the higher molecular weight bands are not resolved. Asterisks represent partial degradation. Background-corrected densitometry profiles are shown for the portions of the gels covering H3 and H4. Gel filtration purified recombinant H3-H4 dimer is shown on the right.

(C) Quantification of the relative molar ratios of H3 to H4 from Coomassie stained gels. Four biological replicates were made. Values are normalised ASF1b. HAT1 is omitted due to histone levels being difficult to discriminate from background staining.

(D) Venn diagram displaying the overlap of interactors associating with the three immunoprecipitated proteins.

(E) Quantification of the relative molar ratios of H3 to H4 from mass spectrometry analysis of three of the biological replicates using label free quantification (LFQ). Values are normalised to ASF1b.
Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures (2013)
Figure 1

Listeria induces a transient activation of host AA starvation pathways. (A) HeLa cells were infected with Listeria for 0.5–4 h, and lysates were analysed by blotting using indicated antibodies. (B) HeLa cells left unstimulated (CTR) or infected with Listeria for 1 or 3 h, analysed by IF using antibodies against mTOR and LAMP2. (C) Percentage of Listeria‐infected cells displaying mTOR localization to LAMP2+ vesicles. Values are means±s.e.m. n=3. ***P0.001 and **P0.01 over uninfected. (D, E) HeLa cells were infected with Listeria for 0.5–4 h, and lysates were analysed by blotting using indicated antibodies. (F) qPCR analysis of ATF3 induction following infection of HeLa cells with Listeria for 1–4 h. Values are means±s.e.m. n=3. Scale bars: 10 μm.Source data for this figure is available on the online supplementary information page.
Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures (2013)
Figure 2

Listeria toxins cause activation of host AA starvation pathways. (A) HeLa cells were infected with Listeria wild‐type (WT) or the LLO/PlcA/PlcB triple knockout (TKO) mutant strain for 0.5–4 h, and lysates were analysed by blotting using indicated antibodies. (B) qPCR analysis of ATF3 and IL‐8 induction following infection of HeLa cells with Listeria WT or TKO for 1–4 h. Values are means±s.e.m. n=3. (C) HeLa cells left unstimulated (CTR) or infected with Listeria TKO for 1 h, analysed by IF using antibodies against mTOR and LAMP2. (D, E) Percentage of cells infected with either WT or TKO Listeria strains displaying one or several LAMP2+ (D) or NDP52+ (E) Listeria vesicles. Values are means±s.e.m. n=3. *P0.05; **P0.01. Scale bars: 10 μm.Source data for this figure is available on the online supplementary information page.
Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures (2013)
Figure 3

Distinct roles of LLO and PlcA/B in the modulation of host AA starvation pathways. (A) qPCR analysis of ATF3 induction following infection of HeLa cells with WT, TKO, PlcA/B− or LLO− Listeria strains for 1–4 h. Values are means±s.e.m. n=3. (B) HeLa cells were infected with WT, PlcA/B− or LLO− Listeria strains for 0.5–4 h, and lysates were analysed by blotting using indicated antibodies. (C) HeLa cells left unstimulated (CTR) or infected with WT, PlcA/B− or LLO− Listeria strains for 1 h, analysed by IF using antibodies against mTOR and LAMP2. The arrow shows a Listeria vacuole positive for mTOR and LAMP2, and arrowheads indicate mTOR‐ and LAMP2‐positive late endosomes or lysosomes. (D, E) Percentage of cells infected with WT, PlcA/B− or LLO− Listeria strains displaying one or several LAMP2+ (D) or NDP52+ (E) Listeria vesicles. Values are means±s.e.m. n=3. **P0.01. (F) Percentage of cells infected with WT Listeria, in the presence (black bars) or absence (white bars) of rapamycin, displaying one or several GFP‐LC3+ Listeria autophagosomes. Values are means±s.e.m. n=2. Scale bars: 5 μm.Source data for this figure is available on the online supplementary information page.
Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures (2013)
Figure 4

PlcA/B‐dependent accumulation of NDP52+ granules in Listeria‐infected cells. (A) HeLa cells left unstimulated (CTR) or infected with Listeria WT for 1–4 h, or with Listeria TKO for 4 h and with analysed by IF using an antibody against NDP52. (B) Higher magnification view of the selected area from (A) showing that NDP52+ granules generally form at the vicinity of cytosolic Listeria. (C) Percentage of cells infected with Listeria WT for 1–4 h, or with Listeria TKO for 4 h, displaying one or several NDP52+ granules. Values are means±s.e.m. n=3. (D) Percentage of cells infected for 4 h with different Listeria mutants as indicated displaying one or several NDP52+ granules. Values are means±s.e.m. n=3. (E) HeLa cells infected with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA for 3 h, analysed by IF using an antibody against NDP52. (F, G) Percentage of cells infected for 3 h with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA displaying one or several NDP52+ granules (F) or bacteria entrapped in NDP52+ autophagosomes (G). Values are means±s.e.m. n=2. Scale bars: 5 μm.
Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures (2013)
Figure 5

NDP52+ granules in Listeria‐infected cells have characteristics of pre‐autophagosomal structures. (A) HeLa cells transfected with GFP‐LC3 and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52. (B) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected with Listeria WT for 4 h were analysed by IF using an antibody against ubiquitinated proteins (Ubi). (C, D) HeLa cells infected with Listeria WT for 4 h analysed by IF using antibodies against NDP52 and WIPI‐2 (C) or Galectin‐8 (Gal‐8) and calreticulin (D). (E) HeLa cells infected with Listeria WT for 4 h, in the absence or presence of Wortmannin added during the last hour of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. Scale bars: 5 μm for all panels except (D) (1.5 μm).
Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures (2013)
Figure 6

Accumulation of PlcA/B‐induced granules as a result of reduced autophagic flux and PI3P depletion. (A) HeLa cells were transfected with GFP‐RFP‐LC3 and either were left uninfected (t=0) or were infected with Listeria WT or PlcA/B− for 1–4 h. GFP+ RFP+ and GFP− RFP+ puncta appear yellow and red, respectively. Percentage of cells infected with Listeria WT or PlcA/B− for 1–4 h displaying GFP− RFP+ red puncta were quantified. Values are means±s.e.m. n=3. **P0.01; ***P0.001. (B) HeLa cells left unstimulated (CTR) or infected with Listeria WT or PlcA/B− for 4 h were lysed and PI3P levels measured by competitive ELISA. (C) HeLa cells transfected with GFP‐PKCδ and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52. The arrow indicates an NDP52+ granule. (D) HeLa cells infected with Listeria PlcA/B− for 3 h, in the absence or presence of Wortmannin added during the last 10 min of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. The arrow indicates an NDP52+ granule. Scale bars: 5 μm.
Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures (2013)
Figure 7

Inhibition of pre‐autophagosomal maturation by Listeria PlcA/B protects Listeria against autophagy in trans. (A, B) HeLa cells infected for 4 h with Listeria WT expressing GFP (WT GFP), Listeria PlcA/B− expressing mCherry (PlcA/B− mCherry), or both Listeria strains at a 1:1 ratio, were analysed by IF using an antibody against NDP52 (A). (B) Quantification of the percentage of cells displaying NDP52+ PlcA/B− mCherry from the experiments described in (A), in cells infected with either PlcA/B− mCherry alone or WT GFP and PlcA/B−mCherry (co‐infection). Values are means±s.e.m. n=3. **P0.01. (C) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected for 4 h with WT GFP or Listeria PlcA/B− Listeria, either separately (Single) or in combination (Mix) were lysed and colony forming units (CFUs) determined on agar plates containing appropriate selection markers. Each point represents the value from individual independent experiments. **P0.01; *P0.05. Scale bars: 2.5 μm.