EMBO/SourceData huggingface dataset:
Figure 3(A) Immunofluorescence of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging. Scale bar, 5μm. (B) Quantitation of nuclear SREBP1 intensity in A. n>=25 cells as in (A) from three different fields. ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001.(G) Western blot analysis of TMEM33, RNF5, SCAP, SREBP1, HMGCR, LDLR, FDFT1, IDI1, and PKM2 in wild type, PKM2 KO, TMEM33 OE (pool), and two TMEM33OE clones (clones #1 and #8) MDA-MB-231 cells. Arrow indicates nuclear SREBP1. N: nucleus and P: precursor.
Figure 4(B) Western blot analysis of PKM2, PKM1, NRF1, TMEM33, RNF5 and SCAP levels in MEF (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course experiment.(C) Co-immunoprecipitation of endogenous SCAP and RNF5 in Flag-tagged TMEM33 stably expressed MCF7 cells. SEC61β is an ER protein, which is found to interact with TMEM33.(H) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells. 293T cells were stably transfected with shCtrl or shRNF5 constructs, then the indicated plasmids were co-transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG-132 treatment for 8 hours. FLAG M2 beads were used for pull-down.
Figure 5(D) qRT-PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells (n=3). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.(G) Cell proliferation of Ctrl and HA-RNF5 OE MCF7 cells measured by MTT assays (n=8). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.
Figure 6(E) Western blot analyses of TMEM33 and RNF5 in NRF1 overexpressed MCF7 clones. (F) Western blot analyses of TMEM33 and RNF5 in shCtrl and shNRF1 PKM2 KO MCF7 cells. ( (G) Co-immunoprecipitation of VCP with FLAG-tagged PKM1/2 in PKM2 KO 293T cells. FLAG-tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T cells for 24 hours, followed by pull-down using FLAG-M2 beads. PKM1, PKM2 and VCP antibodies were used for immunoblotting.
Figure 7(E) Representative H&E and IHC staining images of TMEM33 and SCAP in WT and TMEM33 KO mouse livers. Scale bars, 100µm.(F, G) Significant decrease of total plasma cholesterol (F) and LDL-C (G) levels in TMEM33-/- and +/- mice relative to WT counterparts (n>=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. n.s denotes no significance.
Figure 8(B) Tumor growth curves of PKM2 WT or KO py8119 cell line allografts in systemic PKM2 KO or WT mice (n=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.(E) The growth curves of py8119 allografts treated with vehicle and TEPP-46 (50mg/kg) for 17 days in mice (n=7). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.
Figure 1(A) Electrophoretic analysis (under non-denaturing conditions) of the association between the indicated IκBα species association and reconstituted nucleosome core particles (NCP).
Figure 2(B) Quantification of the IκBα 60 kDa band (upper panels) and the 37 kDa band (lower panels) is shown as average and s.d. of three independent experiments.
Figure 3(D) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells in several 2-month-old WT mice (each # represents a mouse).(G) Pulldown assay using GST-SUMO IκBα protein and chromatin lysates from 2-month-old WT mice villi.
Figure 4(A) Western Blot analysis of the crypt-derived chromatin fractions incubated with soluble lysates from villi or crypts for the indicated times.(F) Western Blot analysis of a cleavage experiment incubating modified H4 peptides (P1-P9) with soluble lysates from villi or crypts in the presence (+) or absence (-) of the commercial protease inhibitors cocktail. Lanes indicated as P correspond to the control peptide without lysate incubation. Notice the electrophoretic shift of peptides incubated with villus lysates (compared with control peptides) suggestive of post-translational modifications or binding to proteins absent from crypt lysates.
Figure 5WB analysis (C) of the indicated genes or proteins in parental or IκBα KO HT29 cells obtained at pre-confluence or 7 days post-confluence. Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed.WB analysis (E) of MUC5AC in HT29 cells treated with the indicated protease inhibitors. Bars represent mean values ± standard deviation from 3 biological replicates performedWB analysis (G) of MUC5AC in HT29 cells transduced with sh-RNAs against trypsin (T1, T2) or chymotrypsin (C1, C2). Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed
Figure 1(B-D) Kaplan-Meier survival plots comparing the survival probability of all melanoma patients (B), BRAF-mutated patients (C) and BRAF wild-type (WT) patients (D) with high- (magenta) and low (black) MCUA mRNA expression.(E-G) Kaplan-Meier survival plots comparing the survival probability of patients of indicated cancer types that are divided into either high- (magenta) or low (black) MCUA mRNA expression. P-values were calculated by using a log-rank test. Abbreviations: KICH = kidney chromophobe; KIRC = kidney renal clear cell carcinoma; KIRP = kidney renal papillary cell carcinoma; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; PAAD = pancreatic adenocarcinoma.
Figure 3(C) Representative images of the migrated stained 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) cells on the lower side of the insert. (D) Quantification of the transwell migration in 1205Lu stable MCUA_KD lines, based on the number of stained cells (n=4 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(E) Representative images of 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm. (F) Quantification of 1205Lu stable MCUA_KD spheroid core size (n=6 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(G) Representative images of WM3734 shCTRL (black frame), shMCU_1 (darker orange frame) and shMCU_2 (lighter orange frame) melanoma spheroids. Live cells are shown in green. Scale bar: 100 µm. (H) Quantification of WM3734 stable MCUA_KD spheroid core size (n=5 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(L) Representative images of 451Lu wild-type (grey frame), 451Lu-BR3 (lighter green frame), WM983B wild-type (grey frame) and WM983B-BR (darker green frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm. (M-N) Quantification of 451Lu versus 451Lu-BR3 (M) and WM983B versus WM983B-BR (N) spheroid core size (n≥9 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).
Figure 5(A-C) Mitochondrial hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using mito-HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (A). Scale bar: 10 µm. Quantification of mito-HyPer ratio in 1205Lu (B) and WM3734 (C) under resting state (1205Lu ─ shCTRL: n=217 cells from 6 biological replicates; shMCU_1: n=197 cells from 7 biological replicates; shMCU_2: n=212 cells from 7 biological replicates; WM3734 ─ shCTRL: n=104 cells from 6 biological replicates; shMCU_1: n=107 cells from 7 biological replicates; shMCU_2: n=114 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.(D-F) Cytosolic hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (D). Scale bar: 10 µm. Quantification of HyPer ratio in 1205Lu (E) and WM3734 (F) under resting state (1205Lu ─ shCTRL: n=232 cells from 6 biological replicates; shMCU_1: n=290 cells from 6 biological replicates; shMCU_2: n=290 cells from 6 biological replicates; WM3734 ─ shCTRL: n=443 cells from 9 biological replicates; shMCU_1: n=297 cells from 9 biological replicates; shMCU_2: n=233 cells from 9 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.(I-K) Mitochondrial glutathione redox potential, measured with mito-Grx1-roGFP2 in 1205Lu and WM3734 with and without stable MCUA_KD. Representative ratiometric images (F385 nm/F475 nm) are shown for all conditions (I). Scale bar: 10 µm. Quantification in 1205Lu (J) and WM3734 (K) under resting state (1205Lu ─ shCTRL: n=231 cells from 10 biological replicates; shMCU_1: n=176 cells from 7 biological replicates; shMCU_2: n=164 cells from 7 biological replicates; WM3734 ─ shCTRL: n=127 cells from 7 biological replicates; shMCU_1: n=142 cells from 7 biological replicates; shMCU_2: n=116 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.
Figure 6(G-I) Mitochondrial ATP, measured using mito-ATEAM in 1205Lu and WM3734 with and without stable MCUA_KD. Exemplary ratiometric images (FRET/CFP) are shown for all conditions (G). Scale bar: 10 µm. Quantification of basal levels in 1205Lu (H) and WM3734 (I) (1205Lu ─ shCTRL: n=141 cells from 14 biological replicates; shMCU_1: n=143 cells from 12 biological replicates; shMCU_2: n=143 cells from 13 biological replicates; WM3734 ─ shCTRL: n=122 cells from 12 biological replicates; shMCU_1: n=103 cells from 12 biological replicates; shMCU_2: n=99 cells from 13 biological replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(J) Exemplary confocal microscope images of the mitochondrial network (blue: DAPI staining of the nucleus; green: TOMM20 staining of mitochondria); scale bar: 50 µm. (K-O) Electron microscopy of mitochondria of stable MCUA_KD cell lines. (K) Exemplary images of stable MCUA_KD cells' mitochondria. Scale bar: 500 nm. (L-O) Quantification of mitochondrial diameter (L and N) and intercristae distance (M and O) of 1205Lu (L and M) and WM3734 (N and O) cells with and without stable MCUA_KD, presented as boxplot. The box presents the 25 %- quartile, median and 75 %-quartile, the X represents the mean and the whiskers the minimum and maximum, outliers are represented as dots (mitochondrial diameter: 1205Lu ─ shCTRL: n=47 technical replicates; shMCU_1: n=44 technical replicates; shMCU_2: n=40 technical replicates; WM3734 ─ shCTRL: n=35 technical replicates; shMCU_1: n=52 technical replicates; shMCU_2: n=38 technical replicates; intercristace distance: 1205Lu ─ shCTRL: n=330 technical replicates; shMCU_1: n=285 technical replicates; shMCU_2: n=255 technical replicates; WM3734 ─ shCTRL: n=352 technical replicates; shMCU_1: n=433 technical replicates; shMCU_2: n=357 technical replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).
Figure 1E. Total RNAs from naïve CD4+ T cells, PHA-stimulated CD4+ T cells and resting memory CD4+ T cells were extracted and proceeded to RT-qPCR to quantitate the relative expression of CBX4. The expression of CBX4 in PHA-stimulated and resting memory CD4+ T cells was normalized to naïve CD4+ T cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3B. Super-resolution SIM image of CBX4 in HEK293T cells. DAPI was used to dye DNA which was colored into blue. FITC-tagged antibodies were used to label endogenous CBX4 which was colored into green. Data information: Scale bars in (B) 5 μm. All the samples were imaged to obtain at least three sets of images.C. The distribution of CBX4 bodies diameters. The black line indicated the median value of diameters which was 512 nm. Data were collected from three cells of three independent samples.F-L. RFP-tagged EZH2, RING1B, SUMO4, SUZ12, CHAF1A, DNMT1 and HP1α were co-overexpressed with GFP-tagged CBX4 in HEK293T cells, respectively. SIM imaging was performed for each combination. The line scan profiles of SIM images which showed co-localization and non-co-localization between CBX4 and its partners were shown on the right of each panel. The red arrows in merged images indicated the positions where the line scans profiled. M-O. GFP-tagged CBX4 was overexpressed in HEK293T cells, followed by treating with AF568-tagged antibodies against H3K4me3, H3K9me3 and H3K27me3, respectively. CBX4 and each histone modification were imaged with SIM. Line scan profiles were shown on the right of each panel. Red arrows in merged images indicated the positions where line scans profiled. Data information: Scale bars 5 μm. All the samples were imaged to obtain at least three sets of images.
Figure 4A. FRAP images of CBX4 bodies to indicate the internal diffusion and diffusion across boundary properties. GFP-tagged CBX4 was overexpressed in HEK293T cells. These live cells were proceeded to time series imaging 24 hours post transfection. Six GFP-CBX4 bodies were circled and marked as region of interest 1 (ROI 1), ROI 2, ROI 3, ROI 4, ROI 5 and ROI 6. Three CBX4 bodies (ROI 4, ROI 5 and ROI 6) were bleached with strong 488 nm laser pulse. Another three unbleached CBX4 bodies (ROI 1, ROI 2 and ROI 3) were set as control. Images were captured every 4 s. The right histogram showed relative fluorescence intensities of unbleached and bleached CBX4 bodies in each time point. Data information: Data represented mean ± SEM in biological triplicate in (A). Scale bars in (A) represented 5 μm. All the samples were imaged to obtain at least three sets of images.P. RFP-tagged EZH2 was co-overexpressed with GFP-tagged CBX4 mutant named CBX4mut which was CBX4-LTVTmut. SIM images were captured. The line scan profile was shown on the right. The red arrow within merged image indicated the line scan position. Data information represented 5 μm. All the samples were imaged to obtain at least three sets of images.
Figure 5G. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO1-EZH2 within each group were showed below the figure. H. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO4-EZH2 within each group were showed below the figure.
Figure 6B. Two micrograms (2 μg) of HA-tagged EZH2 were co-overexpressed with 4 μg of Flag-tagged SUMO4, 250 ng of Flag-tagged UBC9, 250 ng of Flag-tagged CBX4 or CBX4mut within 6 cm dishes. The CBX4mut was the LLPS-deficient CBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO4-EZH2 were marked below the panel.D. The endogenous EZH2 in TZM-bl cells within 6 cm dishes was knocked down by siRNAs targeting 3'UTR of EZH2 mRNA, followed by the overexpression of 2 μg of EZH2, 4 μg of Flag-tagged SUMO4, 250 ng of Flag-tagged UBC9 or 250 ng of Flag-tagged CBX4. EZH2 was IP with anti-EZH2 antibodies. IP samples were IB with anti-EZH2 antibodies. Total samples were IB with anti-EZH2, anti-H3K27me3, anti-Histone H3 and anti-GAPDH antibodies. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.E. In the first group, HEK293T cells within 6 cm dishes were transfected with empty vectors. In the second group, 3 μg of EZH2 was overexpressed. In the third group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED and 2 μg of SUZ12. In the fourth and sixth group, 3 μg of EZH2 was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. In the fifth group, 3 μg of EZH2mut (Y731D) was co-overexpressed with 2 μg of EED, 2 μg of SUZ12, 2 μg of SUMO4, 500 ng of UBC9 and 2 μg of CBX4. Forty-eight hours post transfection, EZH2 and EZH2mut were IP with anti-EZH2, followed by the incubation with 2 μg of mononucleosomes (H3.1) and 20 μM of the cofactor S-adenosyl-L-methionine (SAM) to measure the methyltransferase activity of EZH2. The IP reaction in the sixth group was incubated with 2 μg of mononucleosomes (H3.1 K27M) and 20 μM of SAM. Both total and IP samples were IB with anti-EZH2, GAPDH, H3K27me3 and H3.1 antibodies. The purities of recombinant mononucleosomes which included H3.1, H2B, H2A and H4 were verified by Coomassie blue staining. The expression ratios of SUMO4-EZH2 and H3K27me3 were marked below the panel.
Figure 7A. PHA-activated primary CD4+ T cells were infected with wildtype HIV-1 viruses which harbored a GFP ORF after Nef gene, followed by infecting with shluc and shCBX4 lentiviruses respectively. The percentages of GFP-positive cells, which were HIV-1-expressing cells, were monitored every three days. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.B, C. Primary CD4+ T cells were treated as in (A). On Day 6 post infection with HIV-1 viruses and shRNA lentiviruses, ChIP assays with antibodies against H3K27me3 and H2AK119Ub were conducted in both shluc and shCBX4 groups. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by two-way ANOVA with Sidak's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.D. PHA-activated CD4+ T cells were infected with wildtype HIV-1 viruses as in (A). One group of HIV-1-infected CD4+ T cells were left untreated. One group of HIV-1-infected cells were infected with shluc. Another group of HIV-1-infected cells were infected with shCBX4. Two weeks later, one part of HIV-1-infected CD4+ T cells were reactivated by αCD3/αCD28/IL-2. One part of shluc- and shCBX4-infected cells were co-stimulated with LRA JQ-1. GFP-positive cells in each group were measured by flow cytometry to indicate reactivated HIV-1-infected cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.E-G. Primary CD4+ T cells, which contained latent HIV-1-infected cells, were isolated from three HIV-1-infected individuals. One group of cells were untreated. One group of cells were activated with αCD3/αCD28/IL-2. Another two groups of cells were transfected with siNC and siCBX4 utilizing 4D-Nucleofector System respectively. Three days post transfection, the amounts of intracellular HIV-1 RNAs within each group were quantitated by RT-qPCR and represented as 103 copies per million CD4+ T cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 1Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(B) Dose-response assay using increasing numbers of Nrg1 type I β1a-expressing PC12 cells. For each 96-well, 40,000 split TEV assay cells were co-plated with increasing numbers of Nrg1-expressing cells, and incubated for 24h. Half-maximal activation is reached at 5,000 Nrg1-expressing cells. The Z' factor is 0.5 indicating a large separation band for this assay.Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(C) Adding 10ng/ml EGFld resulted in a two-fold activation. Per 96-well, 40,000 split TEV assay cells were co-plated with empty PC12 cells (no Nrg1 expression), 10,000 Nrg1-expressing cells, and 10,000 Nrg1-expressing cells plus 10ng/ml EGFld.Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(D) Lapatinib antagonizes ERBB4-PIK3R1 signaling in a dose-dependent manner. Per 96-well, 40,000 split TEV assay cells were incubated with increasing amounts of lapatinib, followed by co-plating 10,000 Nrg1 type I β1a expressing cells. The inset depicts the IC 50 value in µM. After compound/stimulus addition, each assay was incubated for 24h. RLU, relative luciferase units; Fluc, firefly luciferase activity (black line); Rluc, Renilla luciferase activity (grey line); n=6; data are shown as mean, error bars represent SEM.