EMBO/soda-nel huggingface dataset:
Inheritance of OCT4 predetermines fate choice in human embryonic stem cells (2018)
Figure 3

Figure 3. Transmission and distribution of maternal OCT4 to daughter cells during cell division. A. Lineage of OCT4 expression dynamics. Mean nuclear OCT4 levels were quantified in individual hESCs continuously for 72 h under undifferentiated conditions. Vertical bars represent individual cells that completed an entire cell cycle lifetime. Thin horizontal bars denote cell division events. Color scale indicates low (black), intermediate (red), and high (white) OCT4 expression levels. B. Differences in OCT4 levels between sister cells, cousin cells, second cousin cells, and randomly paired cells. C. Filmstrip showing distribution of OCT4 to daughter cells during cell division. D. Distribution of OCT4 ratios between sister cells before and after BMP4 treatment. Ratios for both sister cells (r and 1/r) are plotted to emphasize central tendency toward r = 1:1. Differently colored curves represent the distribution of ratios at different time points after division. For time points after 5 minutes, the ratio was determined by first calculating a mean OCT4 level for each sister cell among all previous time points and then calculating the resulting ratio between sisters. E. Proliferating H9-OCT4-mCherry hESCs were imaged for 24 h, fixed, and stained for DNA content with DAPI. The probability density of DAPI ratio (blue) and OCT4-mCherry ratio (red) between sister cells that divided ≤1 h prior to fixation was calculated for 151 cell pairs using a normal kernel function. A two-sample Kolmogorov-Smirnov test was used to determine significance. * P < 0.05, *** P < 0.0005, two-sample Kolmogorov-Smirnov test; ns, not significant.
Inheritance of OCT4 predetermines fate choice in human embryonic stem cells (2018)
Figure 4

Figure 4. OCT4 levels established during cell division predict long-term OCT4 levels. A. Single-cell histories of OCT4 dynamics were classified into 3 time periods. Maternal OCT4 encompasses the levels throughout the lifetime of the mother cell. Inherited OCT4 represents the levels established within 1 h of division. Autonomous OCT4 refers to the OCT4 dynamics during the remaining lifetime of the daughter cell. B. OCT4 mixing as a function of cell history. 30 randomly chosen cells were colored according to their OCT4 expression level at 8 h after division. C. Variance in final OCT4 expression explained over time during maternal, inherited, and autonomous periods of cell history. At each time point, the coefficient of determination (R2) between total OCT4 expression levels and final OCT4 expression at 8 h was calculated. D. Estimate of variance explained by asymmetric distribution of OCT4 to daughter cells. The same R2 over time as in panel C, except that mother-daughter traces were randomly sampled from a set of mother-daughter traces that contained sister-cell pairs (blue line) or only one randomly selected sister cell from each pair (red line). E. Difference in variance explained between red and blue traces in panel D. The black line represents the actual difference; the red line shows a smoothing (moving average = 1.25 h) of the black trace to highlight the overall trend.
VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function (2019)
Figure 1

Figure 1. VAP and VAP-associated protein SCRN1 modulate SV cycling

A. Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP-Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 µm (full size) and 5 µm (zoom).

B. Schematic illustration of the Syt antibody uptake assay: live neurons were stimulated with bicuculline and incubated with primary Syt antibodies, next neurons were fixed and stained with secondary antibodies.

C. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).

D. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N=2, n=288-541 boutons.

E. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal.

F. Pull down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1.

G. Pull down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1.

H. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed >10 enrichment of PSM compared to control.

I. Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 µm (full size) and 5 µm (zoom).

J. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).

K. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone or SCRN1 shRNA with GFP-SCRN1. N=2, n=201-300 boutons.

Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.
VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function (2019)
Figure 2

Figure 2. SCRN1 is recruited to VAP at the ER membrane

A. Localization of GFP-SCRN1 and TagRFP-ER in hippocampal neurons (DIV16-18). Scale bars: 10 µm (bottom panel, full size) and 5 µm (top panel; bottom panel, zoom).

B. Localization of GFP-SCRN1 and ssRFP-KDEL in COS7 cells. Scale bars: 10 µm (full size) and 5 µm (zoom).

C. Hippocampal neurons (DIV16) co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).

D. COS7 cells co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 µm (full size) and 5 µm (zoom).

E. Schematic illustration of SCRN1 recruitment to ER membranes upon increasing VAP levels.

F. COS7 cells co-expressing HA-VAPB with GFP-SCRN2 or GFP-SCRN3. Scale bars: 10 µm (full size) and 5 µm (zoom).
VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function (2019)
Figure 3

Figure 3. VAP-SCRN1 interaction at ER is mediated by a single FFAT-like motif

A. Schematic overview of SCRN1 and VAP constructs. Indicated is if expressed VAP/SCRN1 proteins co-localize in both hippocampal neurons and COS cells, and co-precipitate in pull-down assays.

B. COS7 cells co-expressing GFP-SCRN1 with HA-VAPB-K87D/M89D. Scale bars: 10 µm (full size) and 5 µm (zoom).

C. COS7 cells co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).

D. Hippocampal neurons (DIV16) co-expressing HA-VAPB with GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A. Scale bars: 10 µm (full size) and 5 µm (zoom).

E. Quantifications of SCRN1 recruitment to VAPB-positive structures in COS7 cells (%). Left graph: co-expression of GFP-SCRN1 with HA-VAPB or HA-VAPB-K87D/M89D ( N=2-3, n= 44-46). Right graph: co-expression of HA-VAPB with GFP or GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A (N=2-3, n=41-64).

F. Pull down of HEK293T cells co-expressing Myc-VAPB with GFP, GFP-SCRN1, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP-SCRN1-F153A or GFP-SCRN1-F402A.

G. Sequence alignment of the C-terminal human SCRN1, SCRN2 and SCRN3. Amino acid position three (asterix) in FFAT-like motif (orange) of SCRN1 is not shared in SCRN2 and SCRN3.

Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Chi-square test with post-hoc analysis including Bonferroni correction.
VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function (2019)
Figure 4

Figure 4. VAP and SCRN1 control ER remodeling and presynaptic function

A. Summary of observed phenotypes on SCRN1 recruitment and ER morphology in cells co-expressing indicated SCRN1 and VAP constructs.

B. Live hippocampal neurons (DIV17-18) co-expressing TagRFP-ER with GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Scale bars: 5 µm (full size) and 2 µm (zoom).

C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA or VAPA/B shRNAs. Intact ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads) and impaired an non-dynamic ER structures (arrows) are indicated. Scale bars: 5 µm (full size) and 2 µm (zoom).

D. Schematic illustration of the effect of VAP-SCRN1 interactions on ER structure and dynamics.

E. ER nanostructures visualized with GFP-Sec61β in axons of hippocampal neurons (DIV18) immunostained for α-Tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 µm (full size) and 500 nm (zoom).

F. FRAP experiment of TagRFP-ER in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. Scale bar: 2 µm.

G. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons.

H. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV17-18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A. N=2, n=9 neurons.

I. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons.

J. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T=60 sec in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, VAPA/B shRNAs. N=4, n=6-12 neurons.

Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.
MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol (2023)
Figure 1

Figure 1. Rpn4-mediated proteasome induction represents one arm of the mitoprotein-induced stress response.

A Mitochondrial protein import is under surveillance of the proteasome.

B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths. Yeast cells expressing clogger proteins (cytochrome b2-DHFR and cytochrome b2∆19-DHFR) or cytosolic DHFR for comparison under control of the galactose-inducible promoter were grown to mid-log phase on lactate medium. Ten-fold serial dilutions were dropped on lactate (no induction) or lactate with 0.5% galactose medium (induction). MTS, matrix-targeting signal.

C After expression of the clogger b2-DHFR or the DHFR control, cellular DNA was stained with propidium iodide and DNA content was assessed by flow cytometry. Please note that the first peak shows cells in G1 phase, and the second peak represents cells in the G2 phase.

D Rpn4-driven gene induction was measured using a reporter that expressed the yellow fluorescence protein under control of a PACE element. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with galactose before starting the experiment. (Boos et al., 2019). Data are displayed as mean ± standard deviations from n = 3 independent biological replicates. Significance was assessed using two-sided, paired Student's t-test. P values are indicated as asterisks. * P ≤ 0.05, *** P ≤ 0.001.

E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with 0.5% galactose before samples were harvested and lysed. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.

F Cells of the indicated strains were grown to log phase and diluted in lactate (no induction) or lactate with 0.5% galactose (induction) to 0.1 OD600. Growth was monitored upon constant agitation at 30°C.
MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol (2023)
Figure 2

Fig. 2. Competitive inhibition of mitochondrial protein import in ∆rpn4 cells remodels the cytosolic chaperone system.

A The clogger was expressed in wild type (WT) and ∆rpn4 (∆) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a non-inducing lactate medium. Precursor (pre) and mature (m) forms of the b2∆-DHFR clogger were visualized by Western blotting using a DHFR-specific antibody.

B The clogger was induced for 6 h with 0.5% galactose before cellular proteomes were measured by mass spectrometry. See Materials and Methods for further details.

C 3,550 proteins were measured in all samples of the three replicates.

D Principal component analysis. Presence of Rpn4 and clogger expression caused specific changes in the proteome. The different shapes of the datapoints indicate the three different biological replicates.

E GO Term Analysis was done using the GOrilla tool (http://cbl-gorilla.cs.technion.ac.il/)(Eden, Navon et al., 2009). The absence of Rpn4 caused a broad reduction of the components of the proteasome-ubiquitin system. The number of proteins in the indicated GO Components are stated as "Counts".

F Comparison of the proteome of wild type and ∆rpn4 cells after clogger expression for 6 h. Positions of proteins of the proteasome and chaperone systems are indicated in blue and green, respectively. Please note that Hsp104 and Hsp42 (indicated in magenta) show a higher abundance in ∆rpn4 cells. See Dataset EV1 for details.

G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆rpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.

H. Wild type carrying plasmids for the GAL-induced expression of the clogger and for the constitutive expression (from TPI promoter) of Hsp42 and Hsp104 were grown on lactate medium to mid-log phase and used to inoculate cultures with synthetic lactate (no clogger expression) or lactate plus 0.5% galactose (with clogger). Cells growth was measured over time.

I. The respective strains were grown in lactate medium. Growth was monitored upon constant agitation at 30°C.
MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol (2023)
Figure 3

Fig. 3. Mitoprotein-induced stress induces the formation of MitoStores in the cytosol.

A Cytosolic DHFR (control) or the clogger protein b2-DHFR were expressed in wild type and ∆rpn4 cells for 4.5 h. The distribution of constitutively expressed Hsp104-GFP was visualized by fluorescence microscopy. Diameter of Hsp104-GFP-bound aggregates were quantified. . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates. Significance was assessed using two-sided, paired Student's t-test. P values are indicated as asterisks ** p ≤ 0.01, **** p ≤ 0.0001. See also Fig. EV4A for quantification of the number of aggregates per cell. Scale bars, 5 µm.

B Hsp104-GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures. SN, supernatant representing the non-bound fraction. The signals for Ssa1 and Sod1 are shown for control.

C Hsp104-GFP or DHFR-GFP were co-expressed with clogger or the DHFR control in wild type and ∆rpn4 cells. Clogger expression was induced with 0.5% galactose for 6 h before cells were lysed and the extracts were subjected to nanotrap sepharose chromatography. Purified proteins were analyzed by tandem mass spectrometry. Mitochondrial proteins are highlighted in red, specific chaperones and Rnq1 are indicated in gray or green. The Hsp104 data point was omitted for better scaling of the figure. See Dataset EV2 for details.
MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol (2023)
Figure 5

Fig. 5. Mitochondrial proteins colocalize with Hsp104 in MitoStores.

A The mitochondrial proteins Pdb1, Mam33 and Aim17 were co-expressed for 4.5 h with clogger as fusions with the red fluorescent protein RFP (mCherry). Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4.5 h after induction of the clogger or cytosolic DHFR for control. For quantification of colocalization see Fig. EV5D. Scale bars, 2 µm.

B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17-expressing plasmids and grown to mid-log phase in lactate medium. Clogger expression was induced for 4.5 h with 0.5 % galactose before microscopy was perfomed. Please note that in the absence of Hsp42 and Hsp104, no Aim17-RFP-containing granules are formed. Scale bars, 10 µm.

C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N-terminally truncated version of Mam33-RFP (both under control of GAL promoter) together with constitutively expressed Hsp104-GFP (TPI promoter) . The cells were grown on lactate plus 0.5% galactose-containing medium for 4.5 h. The distribution of the fluorescent proteins was visualized by microscopy. Scale bars, 5 µm.

D Wild type cells expressing Hsp104-GFP were grown on glucose or lactate medium to log phase. Cells were diluted and visualized by life cell imaging. Please note, that in glucose media, Hsp104-GFP is initially dispersed in cells and only aggregates when cells become stationary. In contrast, Hsp104-GFP-containing granules are frequent on lactate medium even at log phase. See movie EV1 for life cell imaging of glucose-grown cells. Scale bars, 10 µm.
MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol (2023)
Figure 6

Fig. 6. MitoStores form in proximity to mitochondria.

A After 4.5 h of galactose-induced expression of clogger and Pdb1-RFP, cells were incubated for 4 h in the absence of galactose. Whereas Hsp104-GFP was distributed throughout the cytosol after this chase period, Pdb1-RFP showed a distribution pattern characteristic for mitochondrial proteins. Scale bars, 2 µm.

B To verify that stored proteins are chased into the mitochondrial matrix, Aim17-GFP11 was coexpressed with clogger for 4.5 h in cells containing mitochondria with Oxa1-GFP1-10. Cells were washed in galactose-free buffer and further incubated for 4 h before fluorescence was measured to quantify the matrix-localized Aim17 proteins. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.

C Clogger was expressed for 4.5 h before colocalization of MitoStores (visualized by Pdb1-RFP) and mitochondria (stained with matrix-residing mito-GFP) was assessed. Scale bars, 2 µm.

D Clogger was expressed for 4.5 h in ∆hsp42 cells before Hsp104-GFP was visualized. Please note that in the absence of Hsp42 no aggregates were detected. Scale bars, 5 µm.

E Clogger and Mam33-HA were co-expressed for 4.5 h in wild type and ∆hsp42 cells. Mitochondria were isolated and subjected to Western blotting to detect mature (m) and precursor (pre) forms of Mam33-HA. The matrix protein Tim16 served as loading control.

F The mitochondria used for E were treated with proteinase K (PK) to remove surface-exposed proteins. Mitochondrial membranes were lysed with NP-40 and soluble (S) and aggregated (P) proteins were separated by centrifugation. T, total.

G To measure the toxicity of clogger expression, clogger proteins were induced for 24 h with lactate medium containing 0.5 % galactose in the mutants indicated. Aliquots were removed and the number of living cells were assessed by a plating assay on glucose-containing plates. The resistance of ∆rpn4 to clogger expression depends on Hsp104 and Hsp42. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.

H Schematic representation of cytosolic MitoStores.
Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion (2020)
Fig. 1.

Establishment of SARS-CoV-2 S protein-mediated cell-cell fusion systema. The emerging timeline for highly pathogenic viruses and the proposed Disease X. b. Schematic representation of SARS-CoV-2 S protein. Its S1 subunit contains NTD (14-305 aa), RBD (319-541 aa), and RBM (437-508 aa). Its S2 subunit contains FP (788-806 aa), HR1 (912-984 aa), HR2 (1163-1213 aa), TM (1214-1237 aa) and CP (1238-1273 aa). c. The formation of syncytium in Huh-7 cells 24 h after SARS-CoV-2 infection, with scale bar of 200 μm. d. Images of SARS-CoV- and SARS-CoV-2 S-mediated cell-cell fusion on 293T/ACE2 cells at 2 h (left) and 24 h (right). e. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on 293T/ACE2 cells at 48 h. f. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on Huh-7 cells at 48 h. Scale bar equals 400 μm in d-f.
Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion (2020)
Fig. 4.

EK1-Lipopeptides showed potent inhibitory activity against SARS-CoV-2 infection.a. Amino acid sequences of the designed peptides EK1 and EK1C. The dotted lines represent E-K salt-bridge with i to i + 3, or i + 4 arrangement. b. SARS-CoV-2 S protein-mediated cell-cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 μm). c. Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell-cell fusion. d. Design diagram of EK1-lipopeptides with cholesterol modification, including EK1C1-EK1C7. e. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell-cell fusion. f. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar).
Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion (2020)
Fig. 5.

EK1C4 broadly and potently inhibited cell-cell fusion and PsV infection mediated by S protein of divergent HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SARS-CoV (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), HCoV-NL63 (e), WIV1 (f), Rs3367 (g) and SHC014 (h). i to o. Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i), MERS-CoV (j), HCoV-OC43(k), HCoV-229E (l), NL63 (m), WIV1 (n) and Rs3367 (o). Experiments were repeated twice, and the data are expressed as means ± SD.