EMBO/soda-nel huggingface dataset:
Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis (2021)
Figure 6

Figure 6. Inhibition of LAM tumorigenesis by targeting histamine metabolism and signaling.

A LC-MS/MS quantification of MIAA in plasma from C57BL/6 female mice with or without Tsc2-deficient 105K tumors grown. The asterisks indicate a significant difference with two-sided t-test (P = 8x10-3; replicates/condition n = 5). Average values are indicated with lilac-colored lines.

B Inhibition of Tsc2-deficient 105K tumor growth with different monotherapies, as indicated in the inset. Asterisks indicate significant reductions relative to vehicle (two-way ANOVA; clorgyline P = 0.015, loratadine P = 0.018, and rasagiline P = 0.049). Each data point represents the mean and SEM.

C Further reduction of Tsc2-deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine, relative to rapamycin alone. Asterisks indicate significant reductions relative to rapamycin alone (two-way ANOVA; clorgyline P = 0.035, loratadine P = 0.045). Each data point represents the mean and SEM.

D Tsc2-deficient 105K tumor weight (g) changes at the end of the rapamycin and rapamycin-combination assays. Asterisks indicate a significant reduction relative to control, as determined by a one-sided t-test (loratadine P = 0.009 and rapamycin P = 3x10-5). Average weight values are indicated with lilac-colored lines.

E Inhibition of Tsc2-deficient 105K tumor growth with Maoa expression depletion using shRNAs. Asterisks indicate significant reductions relative to control pLKO (two-way ANOVA; P = 1x10-3). Each data point represents the mean and SEM.

F Inhibition of Tsc2-deficient 105K tumor growth with Hrh1 expression depletion. Asterisk indicates significant reduction relative to control pLKO (two-way ANOVA; P = 0.035) . Each data point represents the mean and SEM.

G Inhibition of Tsc2-deficient 105K tumor growth with administration of α-methyl-DL-histidine. Asterisk indicates significant reduction relative to vehicle (two-way ANOVA; P = 0.028). Each data point represents the mean and SEM.

H Inhibition of Tsc2-deficient 105K tumor growth by the administration of a low L-histidine concentration mouse diet (0.07% versus 0.49%). Asterisks indicate significant reduction relative to 0.49% L-histidine diet (two-way ANOVA; P = 0.001). Each data point represents the mean and SEM.

I Increase in DLCO over a year in patients treated with rapamycin plus loratadine, relative to those treated with rapamycin alone. Significant difference determined by a two-sided Mann-Whitney test (P = 0.045; number (n) of samples are indicated). Average values are indicated with lilac-colored lines.
Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis (2021)
Figure 7

Figure 7. Molecular and histopathological changes associated with targeting histamine metabolism and signaling.

A Top panels, western blot results of HRH1 and phospho-Ser616 DRP1 expression in vehicle control and single-drug treated tumors. Bottom panels show quantifications; significant differences correspond to one-sided t-test (*P = 0.011 and **P = 2x10-3; tumors/group n = 4). Average values are indicated with lilac-colored lines.

B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone, reduced expression of S6 total in the rapamycin-loratadine combination, and raised phospho-Ser616 DRP1 expression in this combination. Bottom panels show quantifications and significant differences with one-sided t-test (HRH1, clorgyline P = 0.011, loratadine P = 0.049, rasagiline P = 0.021; total S6, loratadine P = 4x10-4; phospho-Ser616 DRP1, loratadine P = 0.037; tumors/group n = 4). Average values are indicated with lilac-colored lines.

C Graph showing the percentages of MitoSOX red-positive Tsc2-deficient 105K cells treated for 24 hours with DMSO or drugs in vitro with 10% FBS complete medium (except for the condition without L-histidine). Clorgyline 1 μM, loratadine 100 nM, rasagiline 1 μM, and rapamycin 20 nM. Differences relative to rapamycin alone were determined by a two-sided t-test (DMSO P = 6x10-5, clorgyline P = 2x10-5, loratadine P = 6x10-4, rasagiline P = 4x10-5; combination with clorgyline P = 1x10-4, loratadine P = 3x10-5, and rasagiline P = 9x10-4; replicates/condition n = 5; assays n = 2). The bars indicate mean ± SD.

D Results of blind histopathological evaluation of Tsc2-deficient 105K tumors treated with vehicle or drugs, in monotherapy or in combination with rapamycin. The histograms show the proportion of defined phenotypic scores: 1+, < 5% of the tumor; 2+, 5-50% of the tumor; and 3+, > 50% of the tumor. Cytological atypia was graded as mild (1+), moderate (2+), or severe (3+). Significant differences relative to rapamycin were determined using Fisher's exact test (epithelioid ***P = 9x10-4 and *P = 0.025; fibrosis, clorgyline and loratadine *P = 0.015, and rasagiline *P = 0.032; glandular, clorgyline *P = 0.030 and rasagiline *P = 0.045; and atypia, loratadine P = 0.010).

E Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with vehicle or monotherapies. Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis, whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology. Scale bars are shown.

F Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with rapamycin alone or rapamycin combinations. With the addition of a second drug to rapamycin, tumors more frequently tended to show glandular differentiation and less atypia. Scale bars are shown.
Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly (2022)
Figure 1

Figure 1. CCT5 is a substrate of DCAF12.

(A) In vitro pull-downs between strep(II)-tagged DDB1-DCAF12 and untagged wild type (WT) CCT5 or CCT5 (1-529) (Δ(Ct)) seen on a Coomassie-stained SDS-PAGE gel.

(B) Titration curves between a fluorescent ATTO488CCT520 degron peptide and TbDDB1-DCAF12 or terbium-coupled streptavidin (Tb-SA) (n=3). Signal originating in the absence of TbDDB1-DCAF12 is unspecific and was subtracted in subsequent experiments.

(C) TR-FRET counter-titration of unlabeled wild type (WT) CCT5 or an unlabeled CCT520 peptide into pre-assembled TbDDB1-DCAF12488 (n=3).

(D) TR-FRET counter-titrations of label-free CCT5 C-terminal peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position and corresponding CCT5 amino acid number.

(E) Counter-titration of unlabeled CCT510 mutant peptides into TbDDB1-DCAF12488 (n=3). Sequences of the peptides and IC50 values for the titrations are listed on the table. Peptides are labeled for their degron position.

Data information: In (B,C,D,E), data is presented as means ± 95% confidence interval (CI). Where indicated, 'n' represents biological replicates.
Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly (2022)
Figure 3

Figure 3. DCAF12 uses a surface pocket to bind the CCT5 di-Glu degron.

(A) A close-up view of the cryo-EM map around the DCAF12 pocket. DCAF12 is shown in light blue as cartoons, with key pocket residues shown as sticks. CCT5 residues are shown as green sticks.

(B) Conservation surface mapping of the DCAF12 WD40 domain. The DCAF12 pocket is annotated for its dimensions and structural components. CCT5 degron residues are shown as green sticks.

(C) LigPlot+ diagram of the interactions between DCAF12 and the CCT5 di-Glu degron (Laskowski & Swindells, 2011). The DCAF12 residues forming hydrogen bonds with CCT5 are shown in blue. DCAF12 residues involved in Van der Waals packing are shown with eyelashes in red. CCT5 residues are shown in green with degron positions in parentheses. Hydrogen bonds and salt bridges are shown as orange and black dashed lines, respectively.

(D) Titration curves between a fluorescent ATTO488CCT520 degron peptide and wild type (WT) or mutant TbDDB1-DCAF12 complexes (n=3). Unspecific signal arising from the Tb-SA label was subtracted.

(E) In vitro ubiquitination of CCT5 by wild type (WT) or mutant CRL4DCAF12 complexes in the presence of ubiquitin, ATP, E1 and E2 enzymes.

Data information: In (D), data is presented as means ± 95% CI. Where indicated, 'n' represents biological replicates.
Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly (2022)
Figure 5

Figure 5. CRL4DCAF12 senses the assembly status of TRiC.

(A) Counter-titration of unlabeled CCT5 or unlabeled TRiC into pre-assembled TbDDB1-DCAF12488 (n=3).

(B) In vitro ubiquitination of monomeric wild type CCT5 or TRiC by CRL4DCAF12 in the presence of ubiquitin, ATP, E1 and E2 enzymes. CCT5, but not TRiC, is modified with poly-ubiquitin (Ubn) chains by CRL4DCAF12. Free CCT5 protein carries an N-terminal strep(II) tag and differs in electrophoretic mobility from untagged CCT5 purified in complex with TRiC.

Data information: In (A), data is presented as means ± 95% CI. Where indicated, 'n' represents biological replicates.

Tables and their legends

Table 1. Cryo-EM data collection, refinement and validation statistics.

DDB1-DCAF12-CCT5

(PDB-8AJM)

(EMDB-15484)

DDB1-DCAF12

(PDB-8AJN)

(EMDB-15485)

Model-to-data fit*

CCmask

CCbox

CCpeaks

CCvolume

0.82

0.85

0.81

0.58

0.74

0.65

* The map was locally sharpened (LocScale).

Table 2. Negative-stain EM data collection, refinement and validation statistics.

(PDB-8AJO)

(EMDB-15486)

0.5736

0.8041

0.3107

0.4072

1) No sharpening performed.

2) Side chains were removed from final model.

Expanded view figure legends
Opposing kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends (2018)
Figure 1

Figure 1 - Mcp1 is required for control of interphase microtubule stability by Klp5/Klp6 but not for its motility.

(A) Interphase microtubules (iMTs) (magenta) in fission yeast grow towards the cell end (i), dwell (ii) then shrink (iii).

(B) Cells expressing fluorescently-tagged α2-tubulin (atb2) were imaged every 5 seconds and the dwell time of ~100 individual iMTs recorded within the final 1.1 µm of the cell for each strain. Red bars signify the mean.

(C) Kymographs showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged microtubules (MT) in the presence (top panels) and absence (bottom panels) of Mcp1. Banding on MT, caused by unequal incorporation of fluorescence, illustrates the force exerted on the MT by continued growth into the cell cortex. Dashed yellow line indicates the cell end.

(D) MT growth speed was calculated from kymographs from control (n=16) and ∆mcp1 cells (n=11), Klp5/Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control (n=44) and ∆mcp1 cells (n=32).

(E) Average intensity of Klp5/Klp6 at the plus ends of iMTs from multiple kymographs of control (n=19) or ∆mcp1 cells (n=14).

(F) Mixing experiment to compare fluorescently-tagged Klp5/Klp6 levels between cells either expressing (blue, closed arrowheads) or deleted (red, open arrowhead) for Mcp1 distinguished by the absence of fluorescently-tagged nuclear envelope protein Cut11 (left panel). Scale bar, 5µm. Box plot (right panel) shows quantitated fluorescence values for nuclear levels of Klp5/Klp6 in control (n=44) and ∆mcp1 cells (n=45) and at the MT plus end in control (n=64) and ∆mcp1 cells (n=35) prior to shrinkage.

Data information: In (E) data are presented as mean ± s.d. * P<0.001, n.s. (non-significant) P>0.05 (Kolmogorov-Smirnov test). In (D) and (F) boxes show the interquartile range with the median represented between the lower and upper quartiles, whiskers show the highest and lowest values.
Opposing kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends (2018)
Figure 2

Figure 2 - Tea2 and Klp5/Klp6/Mcp1 antagonistically control microtubule stability.

(A) Log phase cells expressing fluorescently-tagged MTs were imaged and the levels of either fluorescently-tagged Klp5/Klp6 or Tea2 at the plus ends of growing MTs determined. Measurements (klp5-mNG klp6-mNG, n=305; tea2-GFP, n=359) were plotted against microtubule (MT) length and third order polynomial curves fitted to the data.

(B) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Tea2 kinesin in control cells (top panels) or in the absence of Klp6 (middle panels) or Mcp1 (bottom panels). Dashed yellow line indicates the cell end.

(C) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in (B) for control (n=16), ∆klp6 (n=13) or ∆mcp1 (n=14) cells.

(D) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Klp5/Klp6 in control (top panels, repeated from 1C) or ∆tea2 cells (bottom panels). Dashed yellow line indicates the cell end.

(E) Cells expressing fluorescently-tagged tubulin were imaged every 5 seconds and the dwell time of ~100 individual iMTs for each condition recorded within the final 1.1 µm of the cell. Red bars signify the mean.

(F) The strains in (E) were analysed to determine the position of 20 MT shrinkage events relative to the cell end, which are represented both graphically and as a box plot.

Data information: In (C) data are presented as mean ± s.d. * P<0.001, ** P<0.05, n.s. (non-significant) P>0.05 (Kolmogorov-Smirnov test). In (F) boxes show the interquartile range with the median represented between the lower and upper quartiles, whiskers show the highest and lowest values.
Opposing kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends (2018)
Figure 3

Figure 3 - Tea2 and Tip1 co-localise at the microtubule plus end.

(A) Kymograph (top left panels) showing fluorescently-tagged Tea2 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the position of the cell end. Plots (bottom left panels) show both the relative fluorescence intensity and position for Tea2 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar 2µm. Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 (green) and Tip1 (magenta) over time (top right) and the distance of these pixels from the cell end (bottom right).

(B) Data quantitated from multiple kymographs (n=5) of the type illustrated in (A) to display Tea2 and Tip1 intensity at the MT plus end (left plot) and the position of Tea2 relative to Tip1 (right panel) and relative to initial MT contact with the cell end. Error bars show standard deviation.
Opposing kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends (2018)
Figure 4

Figure 4 - Klp5/Klp6 accumulates behind Tea2 at the plus end until just prior to microtubule catastrophe.

(A) Kymograph (top left panels) showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the cell end. Plots (bottom left panels) show both the fluorescence intensity and position of Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2µm. Intensity of the maximal value pixel at the plus end (top right panel) and its position relative to the cell end (bottom right panel) for both Klp5/Klp6 (green) and Tip1 (magenta) plotted relative to MT shrinkage for this kymograph.

(B) Data quantitated from multiple kymographs (n=7) of the type illustrated in (A) and Fig EV4 to display Klp5/Klp6 and Tip1 levels at the MT plus end (top plot) and the position of Klp5/Klp6 relative to Tip1 (bottom plot) before MT shrinkage.

(C) Data quantitated from multiple kymographs (n=8) of the type illustrated in Fig EV5 where fluorescently-tagged Klp5/Klp6 is co-imaged with fluorescently-tagged Tip1 in the absence of Mcp1 and presented as in (B).

Data information: In (B, C) data are presented as mean ± s.d.
LRRK2 activation controls the repair of damaged endomembranes in macrophages (2020)
Figure 1

Fig. 1. LRRK2 is activated by pathogens that damage phagosomal membranes

(A-D) RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h, Ca WT or Ca YL, and Lm WT or Lm Δhly for 60 min. (A) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry. Data represent the mean ± SEM of three independent biological experiments. One-way ANOVA followed by Dunnett's test compared to WT strains. ns= non-significant; *p ≤0.05; **p ≤0.01. (B) Recruitment of endogenous LRRK2 and EGFP-Rab8A to M. tuberculosis (Mtb) was visualised by confocal microscopy. Scale bar = 5 μm. (C-D) Endogenous LRRK2 and Rab8A recruitment to (C) C. albicans (Ca) and (D) L. monocytogenes (Lm) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.
LRRK2 activation controls the repair of damaged endomembranes in macrophages (2020)
Figure 2

Fig. 2. Endolysosomal damage is sufficient for LRRK2 activation

(A) RAW264.7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot.

(B) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot.

(C) Macrophages were pre-treated with either 1 μM GSK inh or 0.1 μM MLi-2 and then treated with 1 mM LLOMe for 30 min. Rab8A and Rab8A pT72 levels were analysed by Western blot.

(D-F) RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (D) Endogenous LRRK2 and (E) Rab8A recruitment to LAMP-1 positive compartments was visualised by immunofluorescence. Scale bar = 10 μm. (F) Quantification of D-E. Data represents the mean ± SEM of three independent biological experiments.

(G-I) LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of (G) LRRK2 or (I) Rab8A positive vesicles per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.
LRRK2 activation controls the repair of damaged endomembranes in macrophages (2020)
Figure 3

Fig. 3. Rab8A is recruited to damaged lysosomes containing Galectin-3 and CHMP4B in response to calcium signalling

(A) RAW264.7 macrophages were electroporated with EGFP-Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin-3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm.

(B) Macrophages were electroporated with EGFP-Rab8A and RFP-Galectin-3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm.

(C) RAW264.7 macrophages were treated with 1 mM of LLOMe and LRRK2, Rab8A, CHMP4B, Galectin-3 and LC3B foci over time was analysed by high-content imaging. Data shows the mean ± SEM of five biological replicates.

(D-H) RAW264.7 macrophages were pre-treated with 10 μM BAPTA-AM for 1 hr, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (E) Rab8A pT72 phosphorylation was analysed by Western blot. (F) LRRK2 and (G) Rab8A recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (H) Quantification of E and F. (D+H) Data represent the mean ± SEM of two to three independent biological experiments. One-way ANOVA followed by Sidak's multiple comparisons test. ns=non-significant; **p≤0.01, *p≤0.05.
LRRK2 activation controls the repair of damaged endomembranes in macrophages (2020)
Figure 4

Fig. 4. LRRK2 and Rab8A coordinate the recruitment of CHMP4B to damaged endolysosomes

(A-B) RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. (A) CHMP4B and (B) Galectin-3 vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B or Galectin-3 positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.

(C-D) Live cell imaging of LysoTracker positive spots in WT, LRRK2 KO or Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.
LRRK2 activation controls the repair of damaged endomembranes in macrophages (2020)
Figure 5

Fig. 5. Damaged endolysosomes are targeted to lysophagy in the absence of LRRK2 and Rab8A signalling

(A-B) WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (A) LC3B and (B) ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.

(C) Representative images of LC3B/LAMP-1 double positive endolysosomes in control and LLOMe treated macrophages. Scale bar = 10 μm.

(D) Analysis of the % of LC3B/LAMP-1 double positive endolysosomes in LLOMe treated macrophages. Data shows mean ± SEM of three independent biological experiments. *p≤0.05, ***p≤0.001 by Student's t-test.
LRRK2 activation controls the repair of damaged endomembranes in macrophages (2020)
Figure 6

Fig. 6. LRRK2 regulates membrane damage-induced bacterial pathogen intracellular growth

(A-C) WT and LRRK2 KO RAW264.7 macrophages were infected with (A) Mtb for 24 h, (B) Ca for 60 min and (C) Lm for 60 min. Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.

(D-F) WT and LRRK2 KO RAW264.7 macrophages were infected with (D) Mtb WT or Mtb ΔRD1, (E) Ca WT or Ca YL and (F) Lm WT or Lm Δhly. Growth measured for (D) 72 h and (E-F) 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.
LRRK2 activation controls the repair of damaged endomembranes in macrophages (2020)
Figure 7

Fig. 7. Macrophages from PD patients accumulate damaged endolysosomes

Monocyte-derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2-3) or R1441C (Donor 1) LRRK2 mutation were pre-treated with 0.1 μM MLi-2 and treated with 1 mM LLOMe for 30 min.

(A) Rab8A and Rab8A pT72 levels were analysed by Western blot.

(B) Rab8A and Galectin-3 positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns=non-significant, **p≤0.01, by One-way ANOVA followed by Sidak's multiple comparisons test.

Expanded View Figure Legends