EMBO/SourceData huggingface dataset:
Figure 1B. Diluting concentrations of 4D7 to bound full-length 30 amino acid peptide compared to scrambled, C-terminal and N-terminal 11 amino acid peptides as shown in A (n=3 biological replicates).F. Proliferation curves of factor-independent TF-1 TpoR CALRdel61 cells cultured with 2, 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). G Proliferation curves of factor-independent TF-1 TpoR CALRdel52 cells cultured with 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). Data information: bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate, with a one-way ANOVA with Bonferroni correction used to determine statistical significance.
Figure 2B. Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells. C. Similar experiment using TPO-independent TF-1 TpoR CALRdel52 cells at 8 hours. D. Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.F. Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF-1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg/mL 4D7 in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel52 cells.G. Western blot of TpoR immunoprecipitation under non-reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-hour treatment with 20 µg/mL 4D7 but not PBS or 20 µg/mL IgG. CALR monomers & dimers are detectable by polyclonal anti-wild type CALR or anti-mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wildtype CALR protein, asterisk, non-specific bands.
Figure 3B. Graph showing the decreased fold change of CD41+CD61+ megakaryocytes cultured in 4D7 compared to IgG. FACS-sorted CD34+ from myelofibrosis patients with CALR or JAK2 mutation were cultured over 12 days without TPO in the presence of SCF, IL-6 and IL-9. Black columns show JAK2V617F mutation positive samples (n= 4 technical replicates of samples from different patients). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.C. Number of CD41+ megakaryocytes derived from isolated CD34+ progenitors from myelofibrosis patients. Number of CD41/CD61+ cells counted on day 12 using trypan blue exclusion (n=1 technical replicate). D. Summary of fold change reduction of CD41/CD61+ megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type, normalised to IgG (n=11 samples from different patients, with 4 technical replicates per sample). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.E Number of CD41+ megakaryocyte colonies from patient samples after 4D7 treatment. CD34+ from patients with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen-based matrix in presence of 20 µg/mL 4D7 or IgG control (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.F. Representative micrographs showing CD41+ colonies in pink and CD41- colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification. Scale bar indicates 100µm. G. Summary of fold change reduction of CD41+ megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG (n= 4 biological replicates). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.H. Number of megakaryocyte colony forming units grouped according to colony cell number after 4D7 treatment in a mutant CALRins5 sample (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.
Figure 4B. Effect of 4D7 on TPO-dependent megakaryocyte differentiation of healthy CD34+ cells cultured with 20 µg/mL 4D7 or IgG control (n= 2 biological replicates, with 2 technical replicates). Data information: Error bars indicate standard error of mean.C. Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG. CFU-GM, colony forming unit- granulocyte macrophage; BFU-E, blast forming unit-erythroid; CFU-GEMM, colony forming unit granulocyte, erythroid, monocyte, megakaryocyte (n=3 biological replicates). Data information: Error bars indicate standard error of mean.F. Western blot showing signalling in ruxolitinib-resistant TF-1 TpoR CALRdel61 compared to ruxolitinib sensitive TF-1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin as indicated. Ruxolitinib-sensitive cells were non-viable after 16 hours of treatment.G. Comparison of cell growth after DMSO, 20 µg/mL 4D7, IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib-resistant TF-1 TpoR CALRdel61. Cells counted using Trypan Blue exclusion (n=3 biological replicates with 3 technical replicates). H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL 4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates). Data information: Error bars on represent standard deviation. All P values are unpaired Student's unpaired t-test.
Figure 5B. Pharmacokinetic measurements of serum levels of 4D7 in mice after intraperitoneal injection at 0, 1, 24, 48, 72 and 110 hours since administration.D. Kaplan-Meier survival curve of bone marrow engraftment model of TF-1 TpoR CALRdel61 cells showing improved survival of mice treated with 4D7 compared with IgG control commencing 1 week after tail vein injection (n=5 mice per treatment). Data information: For all survival curves the log-rank Mantel-Cox test P value is shown.F. Kaplan-Meier survival curve of TF-1 TpoR CALRdel61 chloroma mice treated with 4D7 or IgG until humane killing due to tumour diameter > 30 mm or ulceration. (n=3 mice per treatment) G. Kaplan-Meier survival curve of NSG mice engrafted with ruxolitinib-resistant TF-1 TpoR CALRdel61 treated with 12.5 mg/kg 4D7 or IgG twice weekly (n=5 and 6 mice for IgG and 4D7 respectively). Data information For all survival curves the log-rank Mantel-Cox test P value is shown.
Figure 1 A. RT-PCR of CNPase (left) and MBP (right) mRNAs expression in MO3.13 precursors under control conditions and following 10-200 μM D-Asp exposure for 3 days. Graphs show quantification of ratio of CNPase, and MBP to L19 Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, left panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control (n=3); A, right panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P<0.05 versus control (n=3  B. Western blotting (left) and densitometric analysis (right) of MBP expression in absence or in presence of 10-200 μΜ D-Asp exposure for 5 days Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); B, one-way ANOVA P=0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control (n=3 C. Cell growth analysis of human MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 1-5 days. The density of M03.13 oligodendrocytes was daily recorded through trypan blue dye exclusion. Mean of daily measurements were recorded. The data of each experimental group were normalized to the density of cells plated at day 0 and expressed as percentage of ctrlday0 Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, for each day, two-tailed Student's t test, *P<0.05 versus control at 3 days (n=5) D. FACS-based cell cycle distribution analysis after PI incorporation of MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 3 days. Representative FACS plots of biological replicates are shown (n=5 independent experimental sessions)  E. FACS-based cell cycle distribution analysis after PI incorporation of rat primary OPC in absence or in presence of 100 μM D-Asp for 1 day. Representative FACS plots of biological replicates are shown (n=3 independent experimental sessions)  F. Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin-594 (a-b and c-d, respectively) in rat primary OPC cultured in absence or in presence of 100 μM D-Asp for 4 days. (e), Single representative MBP++ cell. Scale bars: 50 μm in a-d; 10 μm in e. (f-g); Quantitative analysis of Alexa-594-phalloidin-positive oligodendrocytes (f) or MAG+ and MBP++ oligodendrocytes (g) scored in control and D-Asp exposed cells. Alexa-594-phalloidin-positive oligodendrocytes were scored in 4 categories, according to their morphological complexity. In each category, data were normalized on the total number of oligodendrocytes Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each category two-tailed Student's t test, *P<0.05 versus control (n=3)
Figure 3 A. RT-PCR of CNPase mRNA expression in oligodendrocyte MO3.13 progenitors under control conditions and following 200 μM D-Asp exposure for 3 days, in absence or in presence of 10 μM MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel). Graphs show quantification of ratio of CNPase to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, left, one-way ANOVA P=0.009 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); A, right, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3  B. RT-PCR of MBP mRNA expression in MO3.13 cells under control conditions and following D-Asp exposure for 3 days, in absence or in presence of 10 μΜ MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel) Graphs show quantification of ratio of MBP to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin B, left, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); B, right, one-way ANOVA P=0.0004 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3 C. (a-j); Confocal images displaying the expression of NG2 and MAG proteins in rat primary OPC cultured in presence of D-Asp for 3 days, in absence (a-c) or in presence of 10 μM MK-801 (d-f), or 30 nM YM-244769 (g-i) or 100 nM BED (j-l) C. (m-n); Quantitative analysis of NG2+ and MAG+ cells in rat primary OPC cultured in presence of D-Asp for 3 days, in absence or in presence of 10 μM MK-801, 30 nM YM-244769 or 100 nM BED Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using C, m and C, n, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3) D. RT-PCR of NCX3 mRNA expression under control conditions and following 10-200 μM D-Asp exposure or 100 nM PMA for 3 days Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i D, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3) E. RT-PCR of NCX3 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using E, one-way ANOVA P=0.0083 followed by Tukey's post hoc test, *P<0.05 versus control, ˄P< 0.05 versus D-Asp (n=3), n.s, not significant F. RT-PCR of NCX1 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801. Graphs show quantification of ratio of NCX1 and NCX3 to L19
Figure 7 A. Representative confocal double immunofluorescence images displaying MBP (red) and NF200 (green) distribution and their coexpression (white) in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 20 μm. (j), Densitometric analysis of MBP immunofluorescence signal and (k) quantification of MBP-NF200 colocalized points in the middle corpus callosum of septostriatal sections from control mice, and from mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp Data information The values represent the means ± S.E.M. Level of significance was determined by using in: A, j, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group); A, k, one-way ANOVA P=0.0162 followed by Newman-Keuls post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group  B. Western blot and densitometric analysis of MBP levels in corpus callosum lysates from control and cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively. Data were normalized on the basis of α-tubulin and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i B, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group C. Representative confocal double immunofluorescence images displaying GFAP (green) and Iba1 (red) distribution in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 50 μm. (j-k) Quantification of GFAP+ and Iba1+ cells in the middle corpus callosum of septostriatal sections of control mice and of mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp. Data were normalized to the total cell number (Hoechst signal) and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i C, j-k, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group D. Western blot of GFAP and Iba1 protein levels in corpus callosum lysates obtained from control or cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively Data information The values represent the means ± S.E.M. Level of significance was determined by using i D middle panel, one-way ANOVA P=0.0146 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group); D right panel, one-way ANOVA P= 0.0026 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group)
Figure 8 A. Confocal double immunofluorescence images depicting Olig2+ (a-e) and Olig2+-CC1+ (f-j) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice at 5 weeks, in absence (cpz) or in presence of D-Asp (cpz + D-Asp), and 2.5 weeks after cuprizone withdrawal, in absence (vehicle) or in presence of D-Asp (D-Asp). (k, left panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice, and cuprizone+D-Asp-treated mice at 5 weeks. (k, right panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, vehicle, and D-Asp-treated mice 2.5 weeks after cuprizone withdrawal. Arrows point to Olig2+-CC1+ cells. Arrowheads point to Olig2+-CC1+ cells. Scale bar in a-j: 50 μm Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, k left panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus cpz (n=3 mice for each group). A, k right panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus vehicle (n=3 mice for each group  B. Representative electron microscopy images of middle corpus callosum sections from control mice (a), cuprizone treated-mice for 5 weeks in the absence (b) or in the presence of D-Asp (c), and from mice 2.5 weeks after cuprizone withdrawal in the absence (d, vehicle) or in presence of D-Asp (e). Scale bars in a-e: 1 μ  C. Percentage of myelinated axons in the middle corpus callosum of cuprizone (blue) and cuprizone + D-Asp-treated mice (red) for 5 weeks Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, two-tailed Student's t test, *P<0.05 versus cpz (n=4 mice for each group D. Scatter plot analysis of myelin thickness, expressed as individual G-ratios against axon diameters, after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue circle) or D-Asp-treated mice (red triangle). Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters  E. Histogram analysis displaying the G-ratio values of myelinated fibers across the range of axon diameters. Data represent the mean S.E.M. for at least 100 axons from each group Data information: The values represent the means ± S.E.M. Level of significance was determined by usin E, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group F. Percentage of myelinated axons after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue) or D-Asp- treated mice (red). Abbreviations: DEM, demyelination; REM, remyelination Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group)
Figure 1C, D Two types of TNTs were detected in H1299 (C) and A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin (red) were used for fluorescent staining. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.
Figure 2C, D TNT formation (marked by black triangles and white arrowheads) was imaged in control and shM2 H1299 (C) and shM2 A549 (D) cells by confocal microscopy. Scale bar: 60 μm. Boxed areas are enlarged and shown at the bottom of the corresponding images. E, F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 (E) and shM2 A549 (F) cells. 13 images per group (E) and 10 per group (F) were captured for quantification. Three independent experiments were conducted. G Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performedH,I H1299 cells stably expressing the Ctr or GFP-M2 were cultured in the presence of 20 μM 2-DG for 24 h to induce TNT formation. TNTs were imaged by confocal microscopy (A) and the percentage of cells forming TNTs was quantified (B). n=21, 20 and 19 images were captured from Ctr, GFP-M2#1 and GFP-M2#2, respectively. Three independent experiments were performed. Scale bar: 60 μm. Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed
Figure 3A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in collagen-coated dishes and cultured for 24 h before time-lapse life cell images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel. B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm. C Data information: nuclei were stained with Hoechst 33342 (blue).C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells. The percentage of TNTs containing mitochondria was determined. n=168, 187 and 192 for shCtr, shM2#1 and shM2#2 groups, respectively; three independent experiments. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fE Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in galactose medium or co-cultured through a filter.F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f
Figure 4A Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). Scale bar: 25 μm. Data information: nuclei were stained with Hoechst 33342 (blue).C Down-regulating MICAL2PV enhances the mitochondrial localization of Miro1, Miro2 and KIF5B. Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting. The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β-tubulin protein.