EMBO/SourceData huggingface dataset:
Figure 4Western analysis (C) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. (DMSO (Stat5) n = 13, AG1478 (Stat5) n = 7, Lapatinib (Stat5): n = 4, Gefitinib (Stat5) n = 6, DMSO (Akt) n = 9, AG1478 (Akt) n = 3, Lapatinib (Akt) n = 3, Gefitinib (Akt) n = 3, DMSO (Erk) n = 13, AG1478 (Erk) n = 6, Lapatinib (Erk) n = 4, Gefitinib (Erk) n = 8 ; biological replicates combined from three replicate experiments).densitometric quantification (D) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos (DMSO (Stat5) n = 13, AG1478 (Stat5) n = 7, Lapatinib (Stat5): n = 4, Gefitinib (Stat5) n = 6, DMSO (Akt) n = 9, AG1478 (Akt) n = 3, Lapatinib (Akt) n = 3, Gefitinib (Akt) n = 3, DMSO (Erk) n = 13, AG1478 (Erk) n = 6, Lapatinib (Erk) n = 4, Gefitinib (Erk) n = 8 ; biological replicates combined from three replicate experiments). One-way ANOVA and the Dunnett's multicomparison test (pStat5, pAkt) or non-parametric Kruskal-Wallis with Dunn's multicomparison test (pErk) was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.E-F: Confocal images (E) and quantification (F) of immunofluorescence staining of myosin heavy chain in hearts of 4 dpf zebrafish embryos treated with DMSO or dynasore for 2 days. The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross-sectional area of the ventricles. One dot in the boxplots corresponds to one heart (DMSO (thickness, ejection fraction) n = 11, dynasore (thickness, ejection fraction) n = 12, DMSO (area) n = 15, dynasore (area) n = 14; biological replicates). Unpaired two-tailed T-test was used for statistics. Scale bar 50µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.I-J: Western analysis (I) and densitometric quantification (J) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in E. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos (for each group n = 5; biological replicates). Unpaired two-tailed T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.
Figure 5A-B: Confocal images (A) and quantification (B) of immunofluorescence staining of myosin heavy chain and Stat5b in hearts of 4 dpf zebrafish embryos injected with CRISPR/Cas9 and either control or stat5b-targeting gRNA at one-cell stage. The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross-sectional area of the ventricles. Stat5b staining intensity was also quantified. One dot in the boxplots corresponds to one heart (control gRNA: n = 14, stat5b gRNA n = 13; biological replicates from one of three replicate experiments). Unpaired two-tailed T-test was used for statistics. Scale bar 50µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.quantification of the ejection fraction (D). The 4 dpf embryos were treated as in A. Ejection fraction was quantified from the videos. One dot in the boxplots corresponds to ejection fraction of one heart (for each group n = 22; biological replicates from one of three replicate experiments). Unpaired two-tailed T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.
Figure 6C-D: Immunohistochemical analysis (C) and quantification (D) of STAT5 activation in sections representing normal myocardia or pathological cardiac hypertrophy (n = 3, aortic stenosis; n = 2, idiopathic cardiomyopathy). Phospho-STAT5b staining intensity was quantified. One dot in the boxplot corresponds to one subject (normal: n = 6, hypertrophic n = 13; biological replicates). In the boxplot the central band represents the median, the box the interquartile range and whiskers the whole range of values. Unpaired two-tailed T-test was used for statistics. Scale bar 50µm.
Figure 2B Measurement of 2-NBDG uptake in different cell lines by flow cytometry (n=4 biological replicates).C Quantitative RT-PCR detection of glycolytic gene expression levels in HaCaT cells upon miR-31 overexpression. β-actin was used as a house-keeping control gene (n=3 biological replicates)G and H Glycolysis stress test of HaCaT cells upon UK5099 (G, n=4 biological replicates) or PC siRNA (si-PC, H, n=5 biological replicates) in 1mM glucose condition. ECAR and OCR were normalized to the amount of protein.
Figure 3B Western blot analysis of GLS, GS, GOT1, GOT2, AGC1 in HaCaT cells upon miR-31 overexpression, Tubulin α was used as loading control.C Violin plot of glutamine consumption, glutamate secretion, and cystine consumption of HaCaT cells upon different treatments (n=12 biological replicates).G Cellular and extracellular levels of metabolites were detected by targeted metabolomics using C13 glutamine tracing protocol in a time course (n=3 biological replicates).
Figure 4B Cell viability of HaCaT cell was measured by MTS assay (n=4 biological replicates).C Quantitative RT-PCR detection of Has-miR-31-5p expression levels in HaCaT cells exposed to a range of glucose (n=4 biological replicates). RNU6-1 was used as house control gene.D Western blot analysis of GS in HaCaT cells with miR-31 overexpression under different concentrations of glucose or 2-DG treatment, and Tubulin α was used as a loading control.G and H FACS analysis of Th17 differentiation upon TFB-TBOA treatment (G, n=4 biological replicates) or AOA and iGOT1 treatments (H), n≥3 biological replicates in each group.
Figure 5A Representative picture of in situ hybridization (ISH) staining of miR-31 in skin biopsies of healthy individuals and psoriasis patients (n=5 individuals in each group). Quantification of results showing the average level of miR-31 in skin epidermis (right panel). B, basal layer; S+G, spinous and granular layers. Scale bars: 100 μm.B Representative pictures of Immunohistochemical staining of GLUT1, GLS, and GS in skin biopsies of HC and Ps (n=5 individuals in each group). Scale bars: 200 μm.Disease activity was evaluated by epidermal thickness (E) n=5 mice in Vehicle and n=6 mice in other groups.H FACS analysis of Th17 cells proportion in splenic lymphocytes of mice (n=5 mice in Vehicle and n=6 mice in other groups).I Enzyme-linked immunosorbent assay for the determination of IL-17A levels in serum of mice (n=5 mice in Vehicle and n=6 mice in other groups).J Quantitative RT-PCR detection of mmu-miR-31-5p expression levels in skin of mice (n=5 mice in each group).
Figure 1(A) SPT activity and Western blot (WB) analysis of S1PR1 in S1pr1f/f and S1pr1ECKO endothelial cells after 4-OHT (1μM, 72h) treatment. (n=8/group from 3 independent EC isolations/group; 4 mice/EC isolation).
Figure 2(A,B) (A) WB analysis of ORMDLs, NOGO-B, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and (B) relative ORMDLs quantification (n=7 biological replicates).(C) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of C16:0-ceramide (300nM, 30'), with or without SKI II 1µM, 1h pre-treatment). (D) SPT activity in HUVEC in absence or presence of SEW2871 (300nM, 15') (n=7 biological replicates).(E,F) (E) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of SEW2871 (300nM, 15' and 30') and (F) relative ORMDLs quantification (n=6 biological replicates).(G,H) SPT activity and WB analysis of HUVEC treated with (G) siCTRL and siNOGO (40nM, 72h) (n≥4/group), or with (H) siCTRL and siORMDL1/2/3 (40nM, 72h) (n=4 biological replicates).(I) WB analysis of ORMDLs, P-p38, p38, P-JNK, JNK, P-ERK, ERK, P-AKT, and AKT in HUVEC lysates in absence or presence of the indicated inhibitors, with or without S1P (300nM, 30').
Figure 3(A) WB analysis of ORMDL3 (HA), SPTLC1 and SPTLC2 in HUVEC lysates expressing HA-ORMDL3 and treated with cycloheximide (CHX, 10µM) for the indicated period of time.WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and with or without (C) DMOG (1mM, 1h pre-treatment), (D) Eeyarestatin 1 (EER1, 10µM, 1h pre-treatment),WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') (E) MG132 (10µM, 1h pre-treatment).(F) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A in HUVEC lysates in absence or presence of S1P (300nM, 30'), DMOG (1mM, 1h), EER1 (10µM, 1h), and MG132 (10µM, 1h).(G,H) (G) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A, SPTLC1 and SPTLC2 in HUVEC lysates treated with CHX (10µM) for the indicated period of time, and (H) relative quantification(I) WB analysis for Ubiquitin, HA and GFP of HEK293T transfected with GFP-ubiquitin and with the indicated HA-ORMDL3 plasmid, and immunoprecipitated with HA antibody.
Figure 4SPT activity in S1pr1,3f/f and S1pr1,2ECKO mEC , in absence or presence of S1P (300nM, 30') (B) relative WB analysis for S1PR1, ORMDLs, P-AKT and AKT.(G) WB analysis of ORMDLs and S1PR1 in HUVEC lysates in absence or presence of P-FTY720 (100nM, 30') and
Figure 6(A-C) (A) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-IR (Y1150/1151), IR, P-AKT (S473), AKT, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2') or Insulin (1U/mL, 2') and (B-C) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).(D,E) (D) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2'), and in absence or presence of Myriocin (300nM) for the indicated time and (E) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).
Figure 1A) Maximum intensity projections of pediatric (0-12 months) small intestine showing the distribution of CD56, CD90 and CD24 within the ENS. The first image of each row shows a low magnification overview and an inset depicting the DAPI channel in high magnification. The remaining panels correspond to the high magnification inset. Scale bar in overview images = 200 µm. Scale bar in high magnification panels = 40 µm.(F) RT-qPCR from CD56+CD90+ sorted cells vs control non- ENS cells for representative neuronal and glial markers, confirms high enrichment of both neurons and glial cells (mean±SD, N=4 biological replicates, P * < 0.05, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons).
Figure 2B) Representative plot showing full co-expression of TUBB3 (neuronal marker) and SOX10 (glial marker), on ENS cells (bottom) and only minimal and low-intensity background on non-ENS cells (Top) (N=1 representative sample, for stats see Figure 2C). Intracellular staining was performed on formalin fixed cells from ileum, after pre-staining for the extracellular markers. Selection of the ENS cluster in fixed cells is shown in Appendix Figure S2F.C) Percentage of TUBB3+ and SOX10+ cells in ENS and non-ENS cells (mean±SD, TUBB3: N=4 biological replicates, SOX10: N=3 biological replicates). The single markers TUBB3 and SOX10 were gated vs SSC-A as shown in Appendix Figure S2G.
Figure 3B) The distribution of ENS cells and non-nucleated particles is represented in a CD56/CD90 plot. Left: initial selection of ENS cells. Right: zoom on the ENS cluster, where ENS events are shown as non-nucleated, nucleated, merge. For additional details and stats see Appendix Figure S3E.C) ENS cells and non-nucleated particles are shown in a CD56/CD24 plot, as in Figure 3B. Nucleated and non-nucleated events largely overlap and define the same subclusters in CD56/CD24 and CD56/CD90 plots, although the average staining intensity of the three markers is lower for non-nucleated events. For additional details and stats see Appendix Figure S3E.
Figure 4Confocal images of ENS events sorted as nucleated and non-nucleated, as shown in Figure. 3. (A) Most of the staining belongs to what appears to be non-nucleated (DAPI-) cellular debris (putative neuronal terminations) attached to a nucleated cell (putative glial cell). This is particularly true for TUBB3 and CD24, which show (as expected for neuronal selective markers) a starker distinction than CD56 (also expressed by glial cells). At a very low frequency among sorted ENS cells, we could observe putative neurons as represented in (B), with a more homogenous distribution of the three markers. (C) Examples of sorted Hoechst-negative ENS debris, which are confirmed to be non-nucleated (thus validating our gates on Hoechst 33342-A), but positive for all neuronal markers.
Figure 5C) RT-qPCR for neuronal markers on cells sorted for CD24TIP vs all the other ENS cells, confirms strong neuronal enrichment (mean±SD, N=5 biological replicates, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons).E) Graph showing the proportion of the CD24-based subclusters in the nucleated vs non-nucleated fraction of ganglionic and aganglionic colon (mean±SD, N=4 biological replicates, P *** < 0.001, multiple Student's t test, FDR correction for multiple comparisons). Additional details and stats are shown in Appendix Figure S4E-J.
Figure 6G) Stacked violin plots showing enriched genes in specific neuronal subtypes.
Figure 2A. ShRNA targeting Nrg1 type III were validated in rat purified neurons. Western blot analysis for Nrg1 shows reduction of its expression (pro-protein at 150 KDa) using two different harpins, representative of two independent experiments. Clx is calnexin.B. ShRNA#1 and 2 downregulate Nrg1 type III expression also in myelin-forming Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, as shown by western blot analysis, representative of two independent experiments. Clx is calnexin.C, D. Titration of shRNA #1 (C) and of shRNA #2 (D) LVs (lentiviral vectors) on WT Schwann cell/DRG neuron co-cultures analyzed after 7 days of ascorbic acid treatment, with quantification of the mean number of Mbp positive segments and Schwann cell number. In (C) n= 20 (SCR, Scramble), n= 11 (LV #sh1, 12.5%), n= 16 (LV #sh1, 25%), n= 9 (LV #sh1, 50%) DRG/coverslips from two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test. In (D) n= 14 (SCR), n= 10 (LV #sh2, 12.5%), n= 12 (LV #sh2, 25%), n= 79 (LV #sh2, 50%) DRG/ coverslips, representative of two independent experiments. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.E. Representative confocal images of Mtmr2-/- co-cultures transduced using LV expressing Nrg1 type III shRNA #1, with quantification of the percentage of myelin outfoldings, n= number of DRG/coverslips, n= 9, 382 fibers (SCR), n=9, 368 fibers (LV #sh1, 12.5%), n= 8, 327 fibers (LV #sh1, 25%). Asterisks indicate fibers with myelin outfoldings. Results are mean ± SEM, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test, representative of two independent experiments.E. Western blot analysis of Nrg1 and Akt phosphorylation (S473) shows that activation of Akt decreases following Nrg1 type III downregulation (p-Akt/Akt/Actin, ratio between Mtmr2-/- Scramble-treated and Mtmr2-/- sh#1-treated using 12.5% LV is 1 : 0.85). Each lane is a pool of at least 10 different DRG/coverslips, representative of two independent experiments. Bar in (C, D) is 100 μm; in (E) is 20 μm.
Figure 3A. Titration of rhTACE on wild-type (WT) co-cultures stained for Mbp with quantification of Mbp positive fibers and Schwann cell nuclei: n=18 (NT, not treated), n=6 (5ng/ml), n=20 (10ng/ml), n=7 (50ng/ml) DRGs/covers from two independent experiments, p=0.0053, nonparametric one-way ANOVA, followed by Dunn's post hoc test.B. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 5ng/ml and 10ng/ml rhTACE, with quantification of the percentage of myelin outfoldings; n=10 (NT), n=10 (5ng/ml), n=11 (10ng/ml), DRGs/coverslips. A total of 547, 557, and 571 Mbp fibers were quantified, respectively, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.B. Western blot analysis of lysates from Mtmr2-/- cultures treated using 10ng/ml rhTACE showing Nrg1 expression and Akt activation (S473 phosphorylation). Each lane is a lysate of at least n=10 DRGs/coverslips. In Mtmr2-/- (10ng/ml rhTACE) as compared to Mtmr2-/- (NT), Nrg1(150 KDa)/actin ratio is 0.8:1; Nrg1(65 KDa)/actin ratio is 0.48:1; and p-Akt/Akt/actin ratio is 0.6:1.C. Titration of WT co-cultures using different amounts of Niacin, nicotinic acid, with quantification of Mbp positive fibers and Schwann cellnuclei, n=9 (NT), n=8 (1mM), n=9 (5mM), n=8 (10mM), DRGs/ coverslips. Representative of three independent experiments, p=0.0055, nonparametric one-way ANOVA, followed by Dunn's post hoc test.D. Tace activity was measured from lysates of Niacin-treated cultures, n=7 (KO NT) and n=8 (KO 5mM Niacin) number of independent plates, each containing from 10-15 DRGs plated. Note that Tace activity was similar between WT and Mtmr2-/- cultures, one-tailed Mann Whitney U test, p=0.037.D. Immunohistochemistry and confocal microscopy of Mtmr2-/- cultures treated with 1 mM and 5mM Niacin, with quantification of the percentage of myelin outfoldings; n=31 (NT), n=14 (1mM), and n=29 (5mM) DRGs/coverslips from two independent experiments. A total of 1256, 489, and 1305 fibers, respectively, were quantified, p<0.0001, nonparametric one-way ANOVA, followed by Dunn's post hoc test.E. Niacin treatment of Tace-/- explants does not rescue hypermyelination, with quantification, n=8 DRGs/coverslips per condition, p=0.0056, nonparametric one-way ANOVA, followed by Dunn's post hoc test. Representative of three independent experiments.
Figure 5A. Western blot analysis on lysates from treated and not treated explants (at least 10 DRGs/ coverslips per lane) shows that increased Akt activation (S473 phosphorylation) in Vim-/- explants is restored to normal levels following 5 mM Niacin treatment.A. Niacin treatment of Vim-/- explants rescues hypermyelination, n=10 DRGs/ coverslips per condition, with quantification, representative of three independent experiments, p=0.0048, nonparametric one-way ANOVA, followed by Dunn's post hoc test.B. Niaspan administration (daily i.p. injection of 160mg/Kg Niaspan starting at P15 for 15 days) enhances Tace activity in Vim-/- nerves at P30, n=6 animals per genotype, p=0.0325, one-tailed Mann Whitney U test.B. Niaspan treatment of Vim-/- mice does not affect mouse growth, as the growth rate in WT (n=8, saline; n=6 Niaspan) and Vim-/- (n=10 saline and n=10 Niaspan) mice either saline or Niaspan-treated is not significantly different, Linear Mixed Effect (LME) models.C. Semithin section and g-ratio analyses of sciatic nerves at P30 shows that Niaspan does not alter myelin thickness in WT nerves, whereas restores myelin thickness to normal values in Vim-/- nerves. G-ratio values: WT saline, 0.71 ± 0.003, 1872 fibers, n= 5 animals; WT Niaspan, 0.71 ± 0.004, 1882 fibers, n= 5 animals; Vim-/- saline 0.69 ± 0.004, 2313 fibers, n= 6 animals; Vim-/- Niaspan 0.72 ± 0.01, 1616 fibers, n=5 animals. WT saline as compared to Vim-/- saline, p=0.0588; Vim-/- saline as compared to Vim-/- Niaspan, p=0.0431, repeated measures ANOVA, representative of two independent experiments. The number of myelinated fibers is similar between the 4 groups as shown, p=0.3042, nonparametric one-way ANOVA, followed by Dunn's post hoc test.
Figure 1B-E. Left: At least 150 cells were counted per time point and the number of LD per cell was inspected visually and plotted. The median value of each timepoint is indicated by an orange bar and number. Each panel corresponds to a representative time-course experiment in response to each indicated DNA-damaging agent (out of at least 3 per agent). "unt" = untreated condition. "mock" refers to the culture being treated with DMSO, the dissolving agent for CPT. Used concentrations were 100 µg/mL zeocin, 100 µM CPT, 100 mM HU and 15 µg/mL nocodazole. Right: the same cultures were analysed by cytometry to assess DNA content (1C = unreplicated DNA, 2C = fully replicated DNA) during the time-course to confirm that the used agents were causing cell arrest at the expected phases of the cell cycle.H-K. Cells as described in (G) in which the non-induced condition varied between glycerol (H) or raffinose (I-K), and the cells harboured either a plasmid driving 59 cuts in the genome (H-J) or none (K).
Figure 2B. Exponentially growing S. cerevisiae WT, ste∆ and tag∆ cells were treated with 100 µg/mL zeocin and samples collected at the indicated timepoints for western blot analysis and cytometry The activation of the DDR was monitored following the progressive phosphorylation of its downstream effector kinase Rad53. The unphosphorylated and phosphorylated isoforms of Rad53 are indicated (Rad53 and Rad53-P, respectively). Ponceau staining is shown as a loading control. The percentage of Rad53-P was quantified at each point by dividing the raw signal of the upper band by the total signal in that lane, thus plotted in the graph shown on the right. The plotted values represent the mean value of at least 3 independent experiments and the variation is represented by the SEM. Unpaired t-tests were used to compare the potential differences of the means at each time point. Only the p-value(s) for those being significantly different are indicated. C. Identical to (B) but comparing unloaded WT and WT pre-loaded for 2 h with 0.05% oleate in order to inhibit sterol esterification.D. Non-confluent RPE-1 and Huh-7 human cell lines were either left untreated, or treated for 2 h with the DNA-damaging agents MMS (0.005%) or zeocin (10 µg/mL). Prior to that, cells were pre-loaded with oleate (4 h at 60 µM) to inhibit sterol esterification or treated with 30 µM BSA (mock) as a control. The downstream effector kinase of the DDR CHK2 was monitored by western blot for its activation using a specific antibody against its phosphorylation at Thr68 (P-CHK2). The western blot signal for total CHK2, calnexin and the Ponceau staining are used as loading controls. The bars in the graph show the mean value of the P-CHK2 to CHK2 signals ratio for genotoxins-treated cells while the dots correspond to the individual values out of 3 independent experiments. E. Identical to (D) though only in response to zeocin and by inhibiting sterols with the specific inhibitor avasimibe (5 µM for 2 hours). Plot details as in (D).
Figure 4B. Recombination assay details as in (A) using WT, rad3-102, ste∆ and rad3-102 ste∆ strains.C, D. Pulsed Field Gel Electrophoresis (PFGE) performed on DNA samples prepared from RPE-1 or Huh-7 cells that had been treated (or not) with zeocin and/or oleate (2 hours at 10 µg/mL zeocin; pre-treatments for 4 hours with 60 µM oleate) to evaluate the presence of DSBs under such treatments. Intact DNA molecules remain in the well, while broken molecules migrate into the gel. Given the action of zeocin, in-gel molecules can be subdivided in high-molecular weight fragments (indicated by "HMW") and low-molecular weight ones (LMW). Plugs were prepared at 37°C to prevent artefactual breaking of DNA molecules The bottom graphs provide the percentage of total broken molecules per lane with respect to all molecules in that lane. 3-4 independent experiments are plotted, and their median value indicated by an orange bar. The lateral graphs provide the relative intensity (arbitrary units) of signals associated with in-gel broken molecules. The values corresponding to zeocin-treated samples are displayed in pink, those corresponding to oleate + zeocin-treated samples in orange. For pink and orange curves, the mean value of 3-4 independent experiments is displayed in dark colour, while the SEM is indicated in light colours. Statistical analyses of the difference between treatments was assessed using a t-test.