EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Internalization of triple mutant hAPP in wild type, p75NTR knock-out (KO), ΔDD and C259A hippocampal neurons (15min time point). Counterstaining for p75NTR and DAPI are also shown. Scale bar, 10μm. (B) Kinetics of internalization of triple mutant hAPP in hippocampal neurons from wild type, p75NTR knock-out (KO), ΔDD and C259A mice. Shown is mean ± SEM of percentage internalization of total surface APP (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in vs. KO mutants; **, P<0.01, WT vs. all other genotypes (2-way ANOVA). (C) Linear transformation of hAPP internalization kinetics shown in (B). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes. (D) Kinetics of internalization of p75NTR in wild type hippocampal neurons in the presence or absence of NGF. Shown is mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 ± NGF (2-way ANOVA). (E) Linear transformation of p75NTR internalization kinetics IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes. (F) Internalization of endogenous p75NTR in wild type hippocampal neurons (30min time point). Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm. (G) Kinetics of internalization of p75NTR in hippocampal neurons from wild type, ΔDD and C259A mice. Show are mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in mutants vs. WT (2-way ANOVA). (H) Linear transformation of p75NTR internalization kinetics shown in (G). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes. (I) (A) Co-immunoprecipitation between p75NTR and β-adaptin in hippocampal extracts from 6 month wild type and p75NTR mutant mice as indicated. Results shown are representative from 2 independent experiments. Molecular weights are in kDa. IP: immunoprecipitation; IB: immunoblotting; WCL, whole cell lysate.
Figure 7Representative images of whole-mount preparations of the indicated white adipose tissue depots of 5-6 month old Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG male mice. The photographs were taken at 10x magnification. Scale bar = 100 μm. Quantitation of mGFP and mTomato positive adipocytes from each of the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Adipocytes were counted from four non-overlapping images per depot per mouse.
non-GaMT harbors more MT repair sites where cargos frequently pause and reverse.Figure 3Five representative images of MT repair sites in HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. From left to right, control cell, CLASPs&GCC185 KD cell, CAMSAP2 KD cell, AKAP450 KD cell and Centrinone-B treated cell. Grey: α-tubulin; red: GTP-tubulin. Scale bar: 20 μm. Box-whisker plot presents intensity analysis of MT repair sites in HRPE cells under combinative KDs. (1 representative of 3 independent experiments and n= 20 cells). ***p < 0.001, unpaired t-test. Conventional image (left) and STORM image (right) of MTs and MT repair sites. Grey: α-tubulin; yellow: GTP-tubulin; green: Golgi. Scale bar: 5 μm. MT repair sites on GaMTs and non-GaMTs extracted from C. Red: GaMTs; Green: non-GaMTs; yellow: GTP-tubulin. MT repair sites on GaMTs and non-GaMTs separately presented in detail. White arrows indicates GTP-tubulin labeling sites. Scale bar: 5 μm. Box-whisker plot presents the ratio of MT repair sites on GaMTs and non-GaMTs separately. (1 representative of 3 independent experiments and n= 8 cells). ***p < 0.001, unpaired t-test. Representative time series show one fast cargo moving on a GaMT and slowing down at the MT repaire site. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Representative time series show one cargo moving fast between two MT repair sites and slowing down at the MTrepair sites. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm. Pause (I) and reverse (J) events of cargos on MT repair segment and non-MTs repair segment, respectively. (Data were pooled from 2 independent experiments and n= 22 cells). ***p <0.001, ns, no significant difference, unpaired t-test.
Figure 3Kinetic representation of the ordinary differential equation (ODE) model describing WT RAD51 polymerisation on ssDNA, consisting of five parameters: kp (polymerisation forward rate), ku (unstable reverse rate), kq (quasi-stable reverse rate), ks (stable reverse rate) and KD (protomer-protomer interaction affinity). KD predicts the concentrations of RAD51 polymers of variable length in solution, while kp, ku, kq and ks predict the speed of formation of RAD51 polymers on ssDNA. B. Kinetic representation of the ODE model describing WT RAD51 polymerisation on dsDNA, consisting of four parameters: kp, ku, ks, KD. KD predicts the concentrations of RAD51 polymers of variable length in solution, and kp, ku and ks predict the speed of formation of RAD51 polymers on dsDNA. In panels A and B, purple arrows depict a simplified cartoon representation of RAD51 polymerisation in solution. The model allows for any RAD51 n-mer (1 ≤ n < 16) to associate with any other RAD51 m-mer (1 ≤ m < 16) to form an (m+n)-mer (1 < m + n ≤ 16), and any RAD51 k-mer to dissociate into any combination of n-mers and m-mers (k = n + m). The ssDNA and dsDNA models were calibrated by simultaneously fitting the SPR curves of ssDNA dN-X (dN-8, dN-14, dN-17, dN-50) and dsDNA dN-Xp (dN-5p, dN-8p, dN-11p, dN-50p) using the mode ABC-SMC particles. Mean values ± 1 SD of 3 mode ABC-SMC particles derived from model fits of 3 dN-X and dN-Xp repeats (n = 3). ssDNA kp, ku, kq and ks were fit to the ssDNA SPR curves, and dsDNA kp, ku ks were fit to the dsDNA SPR curves. A single KD was fit to all curves. Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels C and D, the KD was fixed to the mean value identified in Figs. 3 A and B (i.e. KD = 1.14 nM), and the concentration of WT RAD51 monomer concentration was varied accordingly. In panel D, the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % WT RAD51 = [n-mer] * n / [WT RAD51monomer]). [WT RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. It is important to note that the assumed maximum polymer length in solution (16-mer) leads to an overestimation of the concentration of each n-mer in solution, given that RAD51 is likely to form polymers of length greater than 16 at 150 nM, 3 µM and 30 µM WT RAD51 monomer concentration. However, this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration.
Hwa undergoes multi-monoubiquitination.Figure 4A Hwa is ubiquitinated by ZNRF3, but not ZNRF3ΔRING. HEK293T cells were co-transfected with Hwa-Flag and ZNRF3 or ZNRF3ΔRING. The lysates were immunoblotted with Myc, Flag, and α-tubulin antibodies. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments. B Hwa ubiquitination was analyzed under denaturing conditions. HEK293T cells co-transfected with wildtype His-HA-Ubiquitin (His-HA-Ub WT) or lysine-less mutant (K0), Hwa-Flag, and ZNRF3-Myc. The lysates were immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. K0, all lysine sites on ubiquitin were mutated. Blots are representative of 3 independent experiments. C Lysine sites (K) in Hwa protein sequence. Three ubiquitination lysine sites (K178, K179, and K234) identified by MS were indicated in red. Other seven lysine sites were indicated in bold. D Screen of ZNRF3-mediated Hwa ubiquitination sites under denaturing conditions. HEK293T cells were co-transfected with His-HA-Ub, Hwa-Flag or Hwa10KR-Flag, and ZNRF3 or ZNRF3ΔRING. The lysate was immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Hwa10KR-Flag mutant, all lysine sites in Hwa sequence were replaced with arginine. Denaturing conditions: before immunoprecipitation, lysates were denatured by adding SDS to a final concentration at 1% and boiling at 95℃ for 15 min. Blots are representative of 3 independent experiments.
STAT1Y701F mutant counteracts the inhibition of L.Figure 5A Legionella pneumophila growth in unstimulated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded on in the 24-well plates and infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.B Legionella pneumophila growth in IFNβ-treated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded in 24-well plates, treated with 500 U/mL of IFNβ and then infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.
Figure 4A Representative images of ovules from wild-type (Col-0), herk1 anj, lre-5 and fer-4 20 hours after emasculation (HAE) displaying the mature female gametophyte structure. Images presented here are maximum intensity projections from confocal microscopy images across several z-planes of ovules stained as per [70]. Scale bars = 50 µm. B Quantification of H2DCF-DA staining of ROS in ovules from wild-type, herk1 anj, lre-5 and fer-4 plants at 20 HAE. Categories are listed in the legend Ovules dissected from at least five siliques per line. *** p<0.001 (χ-square tests). C Percentage of pollen tubes with normal reception at the female gametophyte (black bars) and displaying overgrowth (grey bars) in wild-type, herk1 anj, lre-5 and fer-4 plants, manually selfed at 20 HAE. Fertilisation events counted from at least three siliques per line. *** p<0.001 (Student's t-test).
a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.f8(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti-CD25 Ab. n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). (b) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with an isotype control Ab or anti-CD25-immunodepleting Abs. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (haematoxylin and eosin (HE) staining) are shown for each genotype and treatment. Scale bars, 2 mm. (c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with an isotype control Ab or an Ab specific to FR4. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). (d) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with isotype control Abs or Abs specific to FR4. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (HE staining) are shown for each genotype and treatment. Scale bars, 2 mm.
Figure 2(A) Heat map representation of RNA-seq results. The p65-dependent genes (total 123; defined as genes up-regulated more than 2-fold via TNF-α treatment and down-regulated more than 0.75-fold via BAY 11-7082 treatment) were classified based on the alteration patterns by the siRNA knockdown of KDM7A, UTX, and both, using the algorithm "HOPACH". (B) Three-dimensional (3D) multidimensional scaling (MDS) plots. ECs in the presence or absence of TNF-α ± BAY, siKDM7A, siUTX, or siKDM7A+siUTX were mapped to the 3D space, based on the expression profiles of the TNF-α-responsive genes, by using classical MDS. Each treated cell is represented by a colored dot. (C-E) Bar plot showing mean mRNA levels of VCAM1, ICAM1, and SELE measured 4 hrs after stimulation of ECs with or without TNF-α ± (siKDM7A+siUTX). Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test. (F) Western blots for VCAM1, ICAM, SELE, and ACTB in lysates from ECs treated with or without TNF-α (4 hrs) ± (siKDM7A+siUTX). (G) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± (siKDM7A+siUTX). Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.
The Golgi apparatus is oriented in response to mechanical cues to maintain cell polarity Left: Representative immunofluorescent staining of GM130 (yellow), F-actin (gray) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h.Figure 5Left: Representative immunofluorescent staining of GM130 (yellow), F-actin (gray) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h. Scale bar: 50 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the front (45°-135°) or the rear (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the front or rear of the cell. Left: Representative immunofluorescent staining of GM130 (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs on the stiffness gradient gel. Scale bar: 20 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the stiff side (45°-135°) or the soft side (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the stiff or soft side. Left: Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm laser irradiation. Middle: Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope. Right: Representative live-cell fluorescence imaging of cells marked with the Golgi (green) and nucleus (blue) on the soft region and the stiff island. Representative time-lapse fluorescence images showing the migration of hRPE cell to the stiff island and the reorientation of Golgi in this process. Scale bar: 20 μm.   Quantification of the percentage of cells having a stiff-end biased Golgi positioning. N=29 cells, replicates are biological.
Figure 4(A Nanoscopy of mitochondria of WT and Mic10-KO cells, as indicated. (A) Mitochondria were immunolabeled for Mic60 and ATPB and visualized by 2D STED nanoscopy. (B) Mitochondria were immunolabeled for TOM20 and ATPB and recorded with 3D STED nanoscopy. Image data were deconvolved. Depicted are cross-sections of a mitochondrion (side view). (C) 3D MINFLUX nanoscopy. Mic10-KO cells were immunolabeled for Mic60 using a directly labeled antibody. Colors encode depth information. (D) 3D SIM of living WT and Mic10-KO cells. Cells were labeled with Mitotracker Green. Images show maximum intensity projections. (E) FIB-SEM of Mic10-KO cells. Shown is a representative reconstructed mitochondrion. Dashed box: Orthoslice view on the smaller mitochondrion. The OM and IBM are displayed together in clear grey, the CM in is shown in blue. (F) 2D STED nanoscopy of mitochondria. Mic19-KO cells were transfected with a plasmid for Mic10-FLAG expression under the control of a tetracycline-inducible promoter. Left panel: Mitochondria from noninduced cell. Right: Mitochondria from induced cell expressing Mic10-FLAG.
Figure 1Models of DSB repair by SDSA and BIR. Schematic of the H-150 assay. Blue boxes depict homologous sequence on one end of the DSB and green boxes depict homology on the other end. A DSB is induced at modified MATa locus (Chr. III). Strand invasion occurs within the "Z" sequence (blue box) and after copying Yα-inc (dashed line) and X sequence (150 bp), the X sequence is used to capture the second end during SDSA. When copying continues to the end of chromosome via BIR (dashed line), an acentric chromosome forms and an unrepaired chromosome segment remains, leading to cell death. Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B). Percentage of BIR products among repair outcomes by 6 hr are shown. (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value in all panels. Viability of indicated strains is shown (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value in all panels. Graph shows repair efficiency among all cells by 6 hrs (left) and represents the sum of DSB repair by BIR and SDSA compared to the uncut parental band at 0 hr (mean ± SD; n = 3). Graph shows repair by BIR and SDSA at 6 hrs after break induction in mutant cells compared to repair products in WT, which are set to 100% (right). Data information: Welch's unpaired t-test was used to determine the p-value in all panels. Schematic of H-150 no-gap assay, where 150 bp of Yα homology on the second end of the break is immediately adjacent to the break site. Representative Southern blots showing DSB repair products in indicated mutants in H-150 no-gap assay. Percentage of BIR product among all repair products by 6 hr are shown. (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value in all panels. Viability of indicated strains (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value in all panels. Graphs showing repair efficiency compared to uncut parental band (left) (mean ± SD; n ≥ 3) or compared to the repair products in WT (right) at 6 hrs after DSB induction. See Fig 1F legend for details. Data information: Welch's unpaired t-test was used to determine the p-value in all panels.
a) SH-SY5Y cells were depleted of WASH1, treated with bafilomycin A1 and examined for LC3-II, tubulin, WASH1 and GAPDH levels.f9(a) SH-SY5Y cells were depleted of WASH1, treated with bafilomycin A1 and examined for LC3-II, tubulin, WASH1 and GAPDH levels. Blots shown are representative of two independent experiments in triplicate. (b) Quantification of the representative experiment in triplicate shown in (a), of two independent experiments. Error bars indicate s.e.m. **P=0.0058 by 1-tailed Student's t-test. (c) WASH1-depleted SH-SY5Y cells were trypsinized, stained with propidium iodide and analysed by flow cytometry. Living cells, not stained with propidium iodide, are shown as a percentage of total cells. The graph depicts a representative experiment in triplicate out of two independent experiments, in which at least 10,000 cells were analysed in each replicate. Error bars indicate s.e.m. *P=0.045 by 1-tailed Student's t-test.
Figure 5WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag-NEK7 were untreated (NT), primed with LPS for 4 h, or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblotting. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below. Co-immunoprecipitation analysis of PLK4 interaction with endogenous Spata2 and CYLD in parental HEK293 cells and HEK293 cells stably expressing GFP-PLK4. iBMDMs stably expressing GFP-PLK4 and parental iBMDMs were unstimulated or stimulated with 0.5 ug/ml LPS for 4h. The PLK4 interaction with endogenous CYLD was analyzed by co-immunoprecipitation with anti-GFP antibody. Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP-ASC, pretreated for 2 days with DMSO and two different PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM), primed with LPS for 4 h, and stimulated with Nigericin for 30 min. GFP-ASC specks were imaged and quantified (>500 cells counted). Scale bar, 10 µm. Immunoblot analysis of the indicated proteins in cell supernatants (Sup) and cell extracts (Cell ext) of LPS-primed and Nigericin-stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM) (E) LPS-primed and Nigericin-stimulated iBMDMs stably expressing a control shRNA or 3 different PLK4-specific shRNAs (F). Immunoblot analysis of active caspase-1 (p20) generation in HEK293 cells that were transfected with inflammasome components (Flag-tagged NLRP3, NEK7, ASC, and Caspase-1) along with EGFP, GFP-tagged PLK4, PLK4 kinase dead mutant (D154A), or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post-transfection.
Figure 2A. Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates (IP) from WT and UPF3B mutant cells expressing endogenously FLAG-tagged UPF1 protein as indicated above each lane. The presence of RNase A during FLAG-IP is indicated above each lane. B. Immunoblots showing levels of proteins (right) in input and IP with normal rabbit IgG (IgG-IP) or antibody targeting EIF4A3 (EIF4A3-IP) from WT and UPF3B mutant cells. C, D. CDF plots of (C) PTC+ and PTC-isoforms, (D) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in WT cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. E, F. CDF plots of (E) PTC+ and PTC-isoforms, (F) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. G, H. CDF plots of UPF3B-dependent (G) and -independent (H) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. I. CDF plots of UPF3B-dependent and UPF3B-independent NMD+ isoforms as compared to NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. J. Bar plot showing average fold change as measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and two independent clones of 3AKO, 3BΔ2BD, 3BKO, and 3DKO cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means.