EMBO/SourceData huggingface dataset:
Figure 5HEK293 cells were transfected with α11.2 with the K1647E mutation, α2δ-1, and β2A plus α-actinin-1 WT or E847K/E851K before cell attached patch recording. Representative single CaV1.2 channel recordings of CaV1.2 K1647E with WT or E847K/E851K α-actinin-1. Mean assemble averages for all experiments for both combinations. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (**p<0.01; unpaired t-test; n = 16 to 30; see Suppl. Fig. 8 and Table 6 for more details).
Figure 6HEK293 cells were transfected with WT α11.2, α2δ-1, and β2A + WT α-actinin-1 and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of NeutrAvidin pull-down samples (from lysate containing 600 μg protein) and total lysate samples containing 20 μg protein. α11.2 was detected with an antibody against its HA tag (top), which is present in all constructs used throughout this work. Pull-down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull-down samples. Immunoblotting for vinculin (middle) and tubulin (bottom) indicated that comparable amounts of these intracellular control proteins were present in lysate samples. Their absence in pull-down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation. Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling of control α11.2 samples lacking α-actinin-1 co-expression (mean set to 100%; see Methods; **p<0.01 two-tailed t-test; n = 7).HEK293 cells were transfected with WT or K1647E mutant α11.2, α2δ-1, and β2A, and WT or E847K/E851K mutant α-actinin-1, or CFP alone as negative control and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of pull-down and lysate samples as in (a). Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling in the WT/WT control (mean set to 100%; see Methods). **p<0.01, ***p < 0.001; one-way ANOVA with Tukey post hoc test; n = 4-5).
Figure 7HEK293 cells were transfected with α11.2, α2δ-1, and β2A before whole cell patch recording in 20 mM Ba2+. Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of -80 mV to the indicated potentials (the voltage protocol is schematized in the upper left corner). Representative current traces upon step depolarizations to the reversal potential (Erev) for 20 ms to obtain movement of the ON-gating charges (Qon), and subsequent to -50 mV for 10 ms to obtain tail currents (Itail). Insets: magnifications of exemplary Qon for CaV1.2 WT, K1647A, Y1649A, I1654A, F1658A and K1662E. Plots of Itail (in this panel corrected for variations in cell capacitance) versus total detectable charge transfer for Qon. Slopes of regression curves are strongly reduced for CaV1.2 K1647A, Y1649A and I1654A versus WT (see Table 7 for more details). Dot plots and means + SEM of Qon (in this panel corrected for variations in cell capacitance; *p<0.05; **p<0.01; one-way ANOVA with Bonferroni post-hoc test; n = 13-18; see Table 7 for more details). The reduction in slope of the regression curve of combined population data for K1647A, Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α-actinin binding to the IQ motif is diminished.
Figure 1.(A-D) EM images of cortical neuropil show an absence of AVs and normal neurite profile in 9-mo-old NTg mouse brains (A, arrowheads outline normal neurites) and a marked accumulation of AVs within enlarged or dystrophic neurites in PS1/APP mice (B, arrowheads outline dystrophic neurite profiles in C) and biopsied brain material from an AD patient (B, inset). At higher magnification (C), AVs include autophagosomes (arrows) and multilamellar bodies (arrowhead). In normal dendrites of PS1/APP mice, multiple AVs are frequently seen (D, arrows).(E and F) LC3 quantification analyzed from immunoblots of LC3-I and LC3-II (top) in prefrontal cortical homogenates from cases of nonaffected (Cont), early stage (preclinical) AD (AD-ES), and moderate AD (AD-MS; E), and from brains of 18-22-mo-old PS1/APP (PA) mice (n = 3; F) compared with nontransgenic (NTg) controls (n = 3; *, P < 0.01). Error bars represent SEM.(G-L) LC3 immunofluorescence in 9-mo-old PS1/APP mice can be seen mainly as puncta in dystrophic dendrites of the cortex (G, arrows) and along adjacent dendrites. LC3 (H, arrows) is strong in dystrophic neurites in the periphery (asterisks) of a thioflavin S-labeled plaque core (H, inset) but is less so in neurites closest (H, arrowheads) to the Aβ deposit. LC3 is diffuse and uniform in neurons of NTg mice (I and J) but is predominantly vesicular and distributed more to the dendrites (arrows) than the cell soma (arrowheads) in 9-mo-old PS1/APP cortex (K and L).
Figure 2.Ultrastructural inspection of brain tissue from PS1/APP mice (A-D) shows that AVs (A and B, arrows) are five times more frequent in the dendrites of 8-wk-old PS1/APP than in those of age-matched NTg mice. The frequency of AVs per EM field (C) and mean number of AVs per EM field (D) within the hippocampal molecular layer (n = 3) are shown.LC3immunoblot and analysis (E) and immunofluorescent labeling (F-K) of the hippocampaldendrites (brackets) in 8-9-wk-old PS1/APP and NTg mice show LC3-II elevation (P < 0.05) in 8-wk-old PS1/APP compared with NTg mice (E). (D) *, P < 0.001. (E) *, P < 0.05. Error bars represent SEM. LC3 immunoreactivity in pyramidal celldendrites is increased in 9-mo-old (F-H) and 9-wk-old (I-K) PS1/APPmice and frequently exhibits a punctate labeling pattern, which is more evident at 9 mo than at 9 wk (H and K, arrows) and is uncommon in NTg mice (F and I). Bars (F, G, I, and J), 20 μm; (H and K), 10 μm.
Figure 3.Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques (A). At higher magnification, anti-PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona (B).PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1/APP animals (C and D)Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM).PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain (E and F) by IEM. Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM). IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD (F).
Figure 4.Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B).(C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top).(D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.
Figure 5.Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least).In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.
Figure 6.Immunolocalization of Aβ in AVs from L/APP cells and PS1/APPbrains and γ-secretase components (PS1 and nicastrin) in L/APP cells. Immunogold localization of Aβ40 (A), Aβ42 (B), PS1 (C), nicastrin (D), and in the absence of primary antibody (E) in L/APP cells grown for 6 h in the absence of Leu and His. (F) Quantification of gold particle frequency in AV or tubulovesicular compartments (TBV), which comprise 27.0 ± 11.0 and 19.2 ± 7.2%, respectively, of the total cell area. Error bars represent SEM.(G and H) IEM followed by silver stain enhancement for Aβ40 was performed in 9-mo-old PS1/APP mice.
Figure 7.(A and B) Ultrastructure of AVs (A1 and A2) and lysosomes in subcellular fractions from serum-deprived L/APP cells (A) and Western blot analysis (B) for LC3-II, rab24, APP, βCTF, PS1 (PS1 amino-terminal fragment), and nicastrin (NCT) in L/APP subcellular fractions. AVs, A1 and A2; L, lysosomes; E, tubulovesicular compartments (Golgi/ER/endosomes); P, postnuclear pellet; C, cytosol; M, mitochondria. Empty lanes in the original blot have been removed from the figure and noted with a white line.(C) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L/APP cells grown in −serum media (left) or −Leu/−His media (right). PNP, postnuclear pellet.(D and E) Proportions of the total recovered cell γ-secretase activity in different subcellular fractions after serum deprivation (D) or in uninduced (+serum) or induced (−Leu/−His) cells (E). Comparison of +serum versus −Leu and −His conditions shows the redistribution of γ-secretase activity from the tubulovesicular to AV fractions after macroautophagic induction. G, Golgi; A, AV.(F) Liquid chromatography mass spectrometry analysis of the fluorogenic substrate after incubation with purified AVs. Selected ion chromatogram corresponding to different cleavage products is displayed. Numbers in graph refer to the cleavage/amino acid site from the Aβ peptide.(G) γ-Secretase activity in AV and lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted (PS KO; BD8) or in which human PS1 was stably transfected into the PS KO blastocysts (hPS1; BD8/hPS1). Numbers on x axis are in minutes.(H) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA. Values are given as means ± SEM (error bars).
Figure 1 EdU-labeled viral DNA forms large foci in ThP1 cells infected by HIV-1. EdU images are shown in red, DAPI images in blue. (A) Left: nucleus of an infected cell at 48 h p.i. displays three large and bright EdU foci (colored arrows), as well as small and dim punctae (grey arrows). Center: Magnified views have colored borders matching the colors of the arrows. 3D images show that EdU foci are located within the nucleoplasm (Movie EV1, Appendix Fig. S2). Right: nucleus of an uninfected cell displays no EdU signal. (B) Graph shows EdU intensity profiles along lines shown in A, with colors matching the corresponding arrows.  (C) Dual-color image of an infected cell with EdU (red) and immunolabeled lamin (grey). Curves to the right plot the intensity profile of EdU and lamin along the dashed blue line. The EdU intensity peaks do not coincide with lamin enrichment. See also Fig. EV1A,B.  (D) Multi-color images of an infected cell showing EdU (red) with CA (yellow) and integrase (green). The colocalization of nuclear EdU foci with CA and IN (arrows) confirms that EdU specifically labels viral DNA and shows the presence of these proteins in vDNA foci. See also Appendix Fig. S3. (E,F) vDNA foci are much larger than the predicted size of single viral genomes.  (E) Left image shows an observed, EdU-labeled vDNA focus. Right: simulations of a single linear or circular 10 Kb long chromatinized DNA polymer chain and corresponding predicted images in diffraction-limited (~300 nm resolution) microscopy. (F) Boxplots show the distribution of sizes (FWHM, full width at half maximum) of n=40 measured vDNA foci (for MOI 100) compared to the sizes predicted for linear (left) and circular (right) chains based on n=100 simulated configurations each. Blue lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. All differences are highly significant (Wilcoxon test data vs. model: ***p ≈ 3 ⋅ 10 − 20, circular vs. linear model: ***p ≈ 10 − 17).  (G) Images of vDNA foci (arrows) in ThP1 cells infected with multiplicities of infection (MOI) 10, 50 and 100. Images for MOI 10 are shown at two different contrast levels to reveal dimmer spots. Scale bars, 3 µm.  (H) Boxplots compare the peak intensities inside individual vDNA foci for MOIs 10, 50 and 100. Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Intensities increase significantly with MOI (Wilcoxon tests: MOI 10 vs. MOI 50: *** p ≈ 5 ⋅ 10 − 16; MOI 50 vs. MOI 100: ***p ≈ 5 ⋅ 10 − 5; MOI 10 vs. MOI 100: ***p ≈ 2 ⋅ 10 − 23). Data from one experiment. A replicate showed similar results.  (I) Histograms show the number of foci per nucleus for MOIs of 10 (blue), 50 (green) and 100 (red).
Figure 2vDNA foci are also foci of vRNA. (A) Dual-color image of an infected ThP1 cell showing the viral DNA (red) and the RNA visualized by RNA-FISH (green). Arrows show foci containing both vDNA and vRNA. The bottom row shows the boxed region magnified, with vDNA and vRNA displayed separately and in combination. See also Appendix Fig. S6.(C) Scatter plot shows the intensities of vDNA and vRNA in colocalizing foci with the Spearman correlation r and associated p-value. In ThP1 cells co-infected with a HIV1-GFP virus and a HIV1-LUC virus, nuclear foci contain mixtures of both viruses. (D) Dual-color image shows RNA-FISH against GFP (red) and RNA-FISH against LUC (green). Arrows show foci containing both GFP vRNA and LUC vRNA. The bottom row shows the boxed region magnified, with GFP vRNA and LUC vRNA displayed separately and in combination. See Appendix Fig. S7.  (F) Scatter plot shows intensities of GFP RNA and LUC RNA in colocalizing foci with the Spearman correlation r and associated p-value.
Figure 3 Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6. (B) Green image shows RNA-FISH against NEAT1. (C) Green image shows immunolabeling of SC35. See also Appendix Figs. S9-14.  (D) Boxplots show Pearson correlations r between vDNA (EdU) and CPSF6, NEAT1 or SC35 in 17-19 regions of interest (ROIs). Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts. Highly significant (p<0.01) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs; highly significant negative correlations between vDNA and NEAT1 are found in 19/19 ROIs, and highly significant positive correlations between vDNA and SC35 are found for 16/17 ROIs. Data are from one experiment. For CPSF6, the experiment was repeated with similar results.  (E) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above.
Figure 4 (A) Image of a ThP1 cell infected with an integration-deficient HIV-1 carrying the mutation D116A in the catalytic site of IN. The EdU-labeled vDNA is shown in red and the vRNA detected by RNA-FISH in green. The nucleus (DAPI) is shown in blue. See Appendix Fig. S12 for a larger image region.  (C) Scatter plot shows vDNA and vRNA intensities in colocalizing vDNA and vRNA clusters with the Spearman correlation r and associated p-value.  (D) ThP1 cells were infected with HIV-1 at an MOI of 20, in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Red: vDNA (EdU). (E) Scatter plots show intensities of vDNA and vRNA in detected vRNA clusters.  (F) Image of a ThP1 cell infected by HIV-1 with EU labeled RNA in absence (left) or presence (right) of nevirapine (NVP, 10 µM). Blue: DAPI staining. Green: vRNA (RNA-FISH). Grey: EU-labeled vRNA. See Appendix Fig. S19 for a larger image region. (G) Intensity profiles of EU and RNA-FISH along the dotted lines in panel F (top).
Figure 5 The left panel shows the timeline of drug exposure experiments. (A-D) Images show vRNA and vDNA in an infected ThP1 cell (MOI 20) for each of four experimental conditions. See Appendix Fig. S15-S17 for images of larger regions. (A,B) ThP1 cells were exposed to NVP for 48 h (A) or 72 h after infection (B), then NVP was washed out, and cells were fixed for click chemistry 24 h later. (C) Cells were exposed to PF74 for 72 h after infection, then fixed for click chemistry. (D) Cells were exposed to NVP for 24 h after infection, then NVP was washed out, and cells were exposed to PF74, before being fixed for click chemistry 24 h later. (E,F,H) Scatter plots show vDNA and vRNA intensities in colocalizing clusters with Spearman correlation r and associated p-values. Replicates of the four experiments yielded similar results. (G) qPCR measures DNA synthesis (left) and nuclear import (right) at 72 h p.i. in absence of drug treatment, or after exposure to NVP or PF74. Bars and error bars define mean and standard deviation, respectively. Samples were analyzed in triplicate and two biological replicates were performed.
Figure 6 The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Percentage of GFP positive cells at 3 and 7 days post-infection analyzed by FACS in absence of EdU. (A) Uninfected control cells. (B) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (C) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (D) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. (E-H) Multicolor images of ThP1 cells infected with a GFP-reporter virus. vRNA (RNA-FISH) is labeled in red, GFP in green, and nuclei (DAPI) in blue. (E) Uninfected control cells. (F) Untreated, infected ThP1 cells fixed at 3 d (72 h) p.i. (G) Infected cells were exposed to NVP for 72 h, then NVP was washed out and cells were cultured for another 96 h and fixed at 7 d p.i. (H) Infected cells were cultured for 72 h p.i., then NVP was washed out and cells were exposed to the nuclear import inhibitor PF74 for another 96 h and fixed at 7 d p.i. The experiment was repeated three times.
Figure 7 The left panel shows the timeline of infection and/or drug exposure experiments. (A-D) Multicolor images of vDNA, vRNA, DAPI and/or GFP in monocyte derived macrophages (MDMs) from two different donors, infected with HIV1. Cells from donor 1 were infected with VSV-G-pseudotyped HIV-1ΔEnv Vpx carrying a GFP reporter (A) and cells from donor 2 were infected with a VSV-G-pseudotyped HIV-1ΔEnv without Vpx (B-D). (A,B) Infected cells were left untreated and fixed at 6 d p.i. (C) Infected cells were treated with 10 µM Nevirapine (NVP) throughout the experiment and fixed at 6 d p.i. (D) Infected cells were exposed to NVP for 3 d p.i., then NVP was washed out and cells were cultured for another 4 d and fixed at 7 d p.i. Arrows in (A-D) show the position of selected vRNA foci. (E) Boxplot shows the size of DNA foci in untreated infected cells from donor 2 imaged at 7 d p.i. (B), measured by the FWHM of n=24 foci. The red line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. (F) Boxplot shows Pearson's r, measuring colocalization of DNA with RNA foci in the same cells (n=20). Center line defines the median, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points.  (G) FACS analysis of Gag positive cells from donor 2 in different conditions. Red: uninfected cells. Blue: untreated infected cells at 7 d p.i. as in B. Orange: cells were treated with NVP for 7 d as in C. Green: infected cells were exposed to NVP for 3 d, then NVP was washed out and cells were cultured for another 4 d as in D. Median of fluorescence intensities (MFI) for each sample are shown in the table.
Figure 1Macrophages were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Immunoblot of co-immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β-actin as loading control.(B) Cells were incubated with 10 µg/mL pepstatin A for 4 h prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 6.(D) Immunoblots of SQSTM1 using antibody to SQSTM1 or β-actin. * p<0.05.
Figure 2Macrophages transduced with non-specific scrambled shRNA (shNS) or TLR8 shRNA (shTLR8) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Cells were lysed and immunoblot performed with antibodies to TLR8 and β-actin.(B) Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images from three donors are shown. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 4. * p<0.05.
Figure 3A-B) Macrophages were treated for 24 h with CL097, ssRNA40 or ssRNA41. (A) Top, qRT-PCR for CYP27B1 after 6 h. Bottom, immunoblots of CYP27B1 using antibody to CYP27B1 or β-actin.(B) Top, qRT-PCR for VDR after 6 h. Bottom, immunoblots of VDR using antibody to VDR or β-actin.(C-D) Macrophages transduced with non-specific scrambled shRNA (shNS), CYP27B1 shRNA (shCYP27B1) or VDR shRNA (shVDR) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (C) Left, immunoblots performed with antibodies to CYP27B1, VDR and β-actin. Right, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.(D) Cells were fixed and permeabilized then stained with antibody to LC3B. Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s.e.m., n = 4.(E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or β-actin. Bar charts are reported as mean ± s.e.m., n = 4. * p<0.05.