EMBO/soda-nel huggingface dataset:
Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish (2020)
Figure 1

Figure 1. OXPHOS super-assembly in zebrafish.

A-D, Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (A, B) Immunodetection of the indicated proteins after BNGE, (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates).

E-H, BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (E, F) Immunodetection of the indicated proteins, (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates).

I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.
Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish (2020)
Figure 2

Figure 2. Blue-DiS proteomics of scaf1+/+ and scaf1-/-.

A, Quantitative data-independent scanning (DiS) mass spectrometry protein profiles for CI, CIII and CIV. Vertical numbers indicate the BNGE gel slices. Left and right profiles correspond to scaf1+/+ and scaf1Δ1/Δ1 animals, respectively. Red heatmap corresponds to the E-score from two proteotypic Scaf1-derived tryptic peptides spanning sequences ascribed to the CIII interacting site (in green) and to the CIV interacting site (in yellow). Thick blue line, marked with an asterisk, indicates the putative proteolytic site in Scaf1.

B, Sequence alignment of Scaf1 protein in mouse and zebrafish. Structural and functional regions previously described in mouse are indicated in shaded grey boxes. Thick blue line indicates the proteolytic processing site in the mouse sequence.
Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish (2020)
Figure 3

Figure 3. Phenotype consequences of Scaf1 loss-of-function.

A, B, Representative images from scaf1+/+ and scaf1-/- (A) female and (B) male adult zebrafish.

c-f, Size of scaf1Δ1/ Δ1 and scaf1Δ2/ Δ2 (scaf1-/-) fish in comparison with their respective scaf1+/+ lines, (C) length and (E) weight of females (Δ1 +/+ n=10, Δ1 -/- n=12, Δ2 +/+ n=24, Δ2 -/- n=18); (D) length and (F) weight of males (Δ1 +/+ n=16, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=23).

G-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines).

L-N, Effect of Scaf1 loss of function on female fertility. (L) Number of eggs per clutch (Δ1 +/+ n=12, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=10). (M) Quantification of mature ovary follicles per ovary section (average of three sections/biological replicate) (Δ1 n=5, Δ2 n=8; same number of animals for homozygous scaf1+/+ and scaf1-/-). (N) Representative images of H&E-stained ovaries. Dotted lines delineate adipose tissue.

Data information: One-way ANOVA. Outliers are shown in grey and were not considered for the statistical analysis. Data are represented as mean ± SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Scale bars = 500 μm.
Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish (2020)
Figure 4

Figure 4. Scaf1 loss-of-function leads to alterations in mitochondrial structure and performance.

A-C, Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (A) Representative images showing mitochondria. (B) Mitochondria size (100-150 mitochondria per biological sample). (C) Cristae lumen width (average of three cristae per mitochondria, 20 mitochondria per biological sample). Different biological replicates are represented with different color tones.

D, E, Mitochondrial DNA copy number per nuclear copy number in muscle in females (D) and males (E) (Δ1 n=6, Δ2 n=6 and same number for their respective controls).

F, Survival curve of 4 days post-fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors (three experimental replicates per biological replicate and three biological replicates).

G, H, Oxygen consumption of 48 hours post-fertilization embryos using the XFe24 Seahorse analyzer, (G) Representative oxygen consumption rate (OCR) profile along time and (H) maximum OCR (Δ1 n=11, Δ2 n=11, n=10, n=11 respectively for their controls).

I, Maximum uncoupled (FCCP) OCR in isolated mitochondria from adult fish (male and females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with the indicated site I [pyruvate (Pyr), glutamate (Glu), malate (Mal)] or site II [succinate (Succ)] substrates.

J, K, Respiratory control ratio (RCR)(State 3/State4) (J) and P/O ratio (K) in isolated mitochondria from adult fish (male and females Δ1 n=4, and same number for their respective controls) with the indicated substrates.

Data information: (B-E, G-H, J-K) Unpaired t-test, (F) two-way ANOVA, Sidak's multiple comparison test. (I) two-way ANOVA, post-hoc Fisher's LSD test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Data are represented as mean ± SD. Scale bars = large image 1 µm, small image 50 nm.
Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish (2020)
Figure 5

Figure 5. Diet-induced recovery of scaf1-/- phenotypes.

Data from females.

A, Representative images of scaf1-/- and scaf1+/+ fish fed with the indicated diets.

B, C, Changes in (B) length and (C) weight over time (Δ1 +/+ n=10, Δ1 -/- n=10, Δ2 +/+ n=10, Δ2 -/- n=12-13.

D-F, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (D) Adipose tissue area per total section area (average of three sections/biological replicate) and (E) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) (Δ1 +/+ n=3, Δ2 n=8). scaf1Δ1 and scaf1Δ2 are represented with circles and squares, respectively. (F) Representative images of ventral fat deposits (dotted lines).

G, Number of eggs per clutch (standard diet Δ1 +/+ n=8, Δ1 -/- n=8, Δ2 +/+ n=7, Δ2 -/- n=12, double diet Δ1 +/+ n=7, Δ1 -/- n=8, Δ2 +/+ n=8, Δ2 -/- n=9).

H, Representative images of H&E-stained ovaries. Black dotted lines outline the ovaries, blue dotted line indicates a mature follicle.

I, Quantification of mature ovary follicles per ovary section (average of three sections/biological sample) (Δ1 n=2-3, Δ2 n=6).

Data information: (B, C) Two-way ANOVA, (D, I) unpaired t-test, (E, G) one-way ANOVA. Data are represented as mean ± SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Scale bars = 500 μm.
Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish (2020)
Figure 6

Figure 6. Lack of recovery of Scaf1-/- phenotypes after high-fat diet.

A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets.

C, D, Length of females (C) and males (D) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).

E, F, Weight of females (E) and males (F) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).

G, number of eggs per clutch (standard diet: Δ1 +/+ n=4, Δ1 -/- n=3, Δ2 +/+ n=3, Δ2 -/- n=2, double diet: Δ1 +/+ n=5, Δ1 -/-, n=5 Δ2 +/+ n=1, Δ2 -/- n=2).

H, Quantification of mature ovary follicles per ovary section in haematoxylin-eosin (H&E) histological sections (average of three sections/biological sample) (Δ1 n=3, Δ2 n=3).

I-L, adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females (I, K) and males (J, L): adipose tissue area (I, J) and adipocyte size (K, L).

Data information: (C-F) two-way ANOVA. (G-L) unpaired t-test. Data are represented as mean ± SD. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish (2020)
Figure 7

Figure 7. Molecular basis for diet-induced recovery of scaf1-/- phenotypes.

A, Immunoblot of the indicated proteins of BNGE from female scaf1+/+ and scaf1-/- whole zebrafish mitochondria for the indicated diet (representative of n=2).

B, Maximum uncoupled (FCCP) oxygen consumption rate in whole zebrafish mitochondria (females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with glutamate (Glu), malate (Mal) and succinate (Succ).

C-G, RNAseq data from scaf1+/+ and scaf1-/- skeletal muscle for the indicated diet (standard diet scaf1+/+ and scaf1-/- n=4, double diet scaf1+/+ n=3, scaf1-/- n=4). (C-F) Volcano plots of differentially expressed genes (DEGs). (C) Comparison between scaf1-/- and scaf1+/+ zebrafish in standard diet. (D) Comparison between scaf1-/- and scaf1+/+ zebrafish in double diet. (E) Comparison of scaf1+/+ zebrafish in double diet and standard diet. (F) Comparison of scaf1-/- zebrafish in double diet and standard diet. In blue, significant DEGs (p-adj < 0.05, log2FC > |1|), in grey not significant DEGs, red circles represent non-significant differentially regulated OXPHOS genes, green circles represent significant differentially regulated OXPHOS genes, purple scaf1 (cox7a2l). (G) Heatmap of metabolic differentially regulated pathways in Gene Set Enrichment Analysis (GSEA) in the indicated comparisons.

(H) Heatmap of differentially regulated growth Hallmarks in GSEA in the indicated comparisons.

Data information: (G-H) White squares padj>0.05, colored squares padj<0.05, in blue downregulated gene sets, in red upregulated gene sets. (B) T-test analysis. Data are represented as mean ± SD. * p<0.05, *** p<0.001.

EXPANDED VIEW FIGURE LEGENDS
Neurexins cluster Ca2+ channels within the presynaptic active zone (2020)
Figure 1

Figure 1. Neurexins are not required for synapse formation at the calyx of Held

A. Diagram of the calyx of Held synapse.

B. Strategy for selective deletion of all neurexins at the Calyx of Held by crossing PV-Cre driver mice with neurexin-1/2/3 (Nrxn123) triple conditional knockout (TKO) mice (Zhang et al., 2016; Chen et al., 2017).

C. Representative images of calyx synapses from littermate control and Nrxn123 TKO mice. Brainstem sections were labeled with antibodies to vGluT1 (red) and Syt2 (green). Scale bar, 10 μm.

D. Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity (normalized to control).

E-H. Representative traces of spontaneous EPSCs (sEPSCs)(E), and summary graphs of the sEPSC frequency (F), amplitude (G), and kinetics (H) recorded from littermate control and Nrxn123 TKO mice.

Data information: Data in (D, F-H) are means ± SEM with individual data points. Number of image sections/mice (D) or of cells from at least three mice per group (F-H) analyzed are indicated in the bars. No statistical differences were found by Student's t-test.
Neurexins cluster Ca2+ channels within the presynaptic active zone (2020)
Figure 2

Figure 2. Pan-neurexin deletion severely impairs evoked synaptic transmission at calyx synapses All experiments were carried out in acute MNTB slices from littermate control and Nrxn123 TKO mice at P12-14. EPSCs were evoked by afferent fiber stimulation and recorded in postsynaptic whole-cell patch clamp mode.

A. Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s, recorded in a standard bath solution containing 2 mM Ca2+.

B. Pan-neurexin deletion suppresses synaptic transmission. Summary graphs show the amplitude (left) and charge transfer (right) of the first EPSC recorded in response to the paired stimuli.

C. Pan-neurexin deletion more than doubles the paired-pulse ratio (PPR), suggesting a large decrease in release probability.

D. Pan-neurexin deletion decelerates the EPSC time course. Summary graphs show the rise time (left) and decay time constants (right) of the first EPSC in response to the paired stimuli.

E-F. Pan-neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high-frequency stimulus train (100 Hz for 0.5 sec). Left, representative ESPC traces; right, cumulative summary plot of the EPSC amplitudes during the train (dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back-extrapolation to zero time, which is used to correct for vesicle replenishment during the train).

G. Pan-neurexin deletion does not decrease the readily-releasable pool of vesicles, but lowers the initial release probability during a high-frequency stimulus train. Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily-releasable pool size (left), and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability (right).

Data information: Data are means ± SEM with individual data points. Number of cells (from at least three mice per group) analyzed are indicated in the bars (B, D, and G) or in the graph (F) and apply to all graphs in a series; statistical differences were assessed by Student's t-test (**P < 0.01;***P < 0.001; non-significant differences are not marked).
Neurexins cluster Ca2+ channels within the presynaptic active zone (2020)
Figure 4

Figure 4. Deletion of all neurexins from the calyx of Held synapse does not alter the size or properties of presynaptic Ca2+-currents

A. Example traces of presynaptic Ca2+-currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV.

B. Summary graphs of the I-V relationship for the peak Ca2+-current amplitude (left) and of the peak Ca2+-current density (right).

C. Example traces (left) and summary graphs (right) of the Ca2+-current activation and deactivation kinetics. Currents were induced by a 50 ms step depolarization from -80 mV to +10 mV.

D. Example traces of Ca2+-currents evoked by an action potential-equivalent depolarization (APe, from -80 mV to +17 mV for 1 ms; left), and summary graph of the APe-evoked Ca2+-current peak amplitude and charge (right).

E. Example traces of presynaptic Ca2+-currents induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q-type Ca2+ channel blocker.

F. Summary graph of the relative contribution of P/Q-type Ca2+ channels to the total presynaptic Ca2+ currents, as quantified by the relative reduction in Ca2+ currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.

Data information: Data in (B-D, F) are means ± SEM. Number of cells (from three mice per group) analyzed are indicated in the graph (B) or in the bars (C, D and F); no statistically significant differences were observed as assessed by Student's t-test.
Neurexins cluster Ca2+ channels within the presynaptic active zone (2020)
Figure 6

Figure 6. Pan-neurexin deletion depletes K+-currents carried by presynaptic BK-channels in the calyx of Held synapse

A. Representative traces of Ca2+-currents and 4-AP insensitive K+-currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12-P14.

B. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion.

C. Representative traces of presynaptic BK currents, which are calculated by subtracting currents shown in (B) from currents shown in (A).

D. Summary graphs of the capacitance, Ca2+-current density, and BK-current density.

Data information: Data are means ± SEM with individual data points. Number of cells (from at least three mice per group) analyzed are indicated in the bars (D); Statistical differences were assessed by Student's t test. (**P < 0.01)
Neurexins cluster Ca2+ channels within the presynaptic active zone (2020)
Figure 7

Figure 7. Pan-neurexin deletion depletes CaV2.1-type Ca2+-channels and Bassoon from the presynaptic active zone of the calyx of Held

A. Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 (green), CaV2.1-type Ca2+-channels (red), and the active zone protein Bassoon (purple). Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12-14. Scale bar, 10 μm.

B. Summary graphs of the vGluT1, CaV2.1, and Bassoon immunostaining intensity (normalized to control; CaV2.1 and Bassoon signals were quantified over the vGluT1-positive area to measure active zone-localized proteins).

C. Summary graphs of the diameter and total size of the vGluT1-positive presynaptic area of the calyx of Held.

Data information: Data are means ± SEM with individual data points. Number of brain sections and mice analyzed are indicated in the bars (B and C); statistical differences were assessed by Student's t-test (***P < 0.001).
Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy (2012)
figf1

(a) Ba/F3 cells expressing myrPI(3)K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3)K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate. (b) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ± s.e.m. normalized for B2M (n=4).
*P0.05,
**P0.01. (c) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4). (d) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ±  s.e.m. values normalized for B2M (n=3).
**P0.01. (e) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3). (f) Wild-type Ba/F3 cells were cytokine starved overnight and stimulated with mIL-3. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4). (g) Wild-type Ba/F3 cells were incubated with LY294002 in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3). (h)
FOXO1,3,4−/− MEFs and wild-type MEFs were incubated with LY294002 (10 μM) for the indicated times. Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR. Data are represented as mean values normalized for B2M (n=2). Uncropped images of blots are shown in Supplementary Fig. S7.
Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy (2012)
figf2

(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and actinomycin D (1 μg ml−1) or dimethylsulphoxide (DMSO) as a control in the presence of mIL-3 for 16 h. Cell lysates were analysed for protein levels of glutamine synthetase (GS), p27 and actin. Shown are representative blots (n=3). (b) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO-binding sites were transfected in HEK293 cells together with Renilla and FOXO3(A3) as indicated. Luciferase activity was measured 40 h after transfection. Data are depicted as relative luciferase units (RLU) compared with the control. Shown are mean ± s.e.m. values (n=3). (c) HEK293 cells were stimulated with LY294002 for 24 h and chromatin immunoprecipitations were performed. Protein–DNA complexes were formaldehyde-crosslinked, and chromatin fragments from these cells were subjected to immunoprecipitation with a control antibody or antibodies against FOXO3a, as indicated. After crosslink reversal, the co-immunoprecipitated DNA was amplified by rtPCR. Shown are the means of duplicates within one experiment. IP, immunoprecipitate; TSS, transcription start site. (d) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=4). (e) The osteosarcoma cell line U2OS expressing FOXO(A3)-ER was stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin. (f) MSCs expressing FOXO(A3)-ER were stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin. Shown are representative blots (n=3). (g) Wild-type, daf-2 and daf-16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity. (h) Wild-type N2 worms or daf-2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF-16 double-stranded RNA. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity. (g,h) Shown are the means of five independent plates for each condition. Uncropped images of blots are shown in Supplementary Fig. S7.
Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy (2012)
figf3

(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for glutamine synthetase (GS) activity in an enzyme assay (left). In addition, the expression of glutamine synthetase, p27 and actin was determined using western blot (right). Shown are the mean ± s.e.m. (n=3) and representative blots of these experiments.
**P0.01. (b) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT for 16 h in the presence of mIL-3 together with MSO, as indicated. Cell lysates were analysed for glutamine synthetase activity in an enzyme assay (left). Furthermore the expression levels of glutamine synthetase, p27 and actin were determined using western blot (right). Shown are the mean ±  s.e.m. (n=3) and representative blots of these experiments.
*P0.05,
**P0.01. (c) Ba/F3 cells expressing myrPI(3)K-ER were cytokine-starved overnight. The next day, cells were washed in PBS and put in a medium without serum, with or without 4-OHT. At the times indicated, medium samples were taken and analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho-PKB (S473), phospho-FOXO3 (T32) and actin (inset). Shown are the mean of relative amino acid levels, compared with
t=0 (n=2), and representative blots of these experiments. (d) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in serum-free medium containing mIL-3. The medium was analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and analysed for protein levels of glutamine synthetase, p27 and actin (inset). Shown are the mean ±  s.e.m. of relative amino acid levels, compared with
t=0 (n=4), and representative blots of these experiments.
**P0.01. Uncropped images of blots are shown in Supplementary Fig. S7.