Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area ( µm2 ) of DCX + cells was calculated and related to the total area of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Quantification of the number of neuronal DCX + cells by FACS sorting on 60 days old dissociated hCOs . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

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Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d30 fused - organoid ; ventral GFP - f - CTRL and dorsal UL1 .

Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.


Micrographs of sections from d30 fused organoids , showing ventral GFP + cells in the dorsal area . The migrating GFP + cells were counted starting from 100 µm far from the ventral / dorsal border . Quantification of ventrally - derived GFP + cells in dorsal / thickness of the counted area ( µm3 ) . The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig . EV5D . Every dot represents a different hCOs and different areas were counted for each hCO . Nuclei ( blue ) are stained with DAPI .

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Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.


Volcano plot representing proteins identified by high - resolution nanoLC MS / MS analysis . Up - and down - regulated proteins ( - 0 . 3 ≥ log2FC ≥ 0 . 3 ) in human cerebral organoids ( UL1 vs b - CTRL ) are shown in red and green , respectively .

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Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.


A ) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks ( GW ) , 10 GW , and 12 GW and in the adult human thyroid : quantitative PCR results normalized to one of three thyroid tissues at 8 GW value ( in black ) and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus 8 GW ( in black ) using ANOVA test showed no significant

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A) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW, and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differences


B ) Tubb1 is expressed in the developing mouse thyroid at E13 . 5 , E15 . 5 , and E17 . 5 and in the adult mouse thyroid : quantitative PCR results normalized to one of three thyroid tissues at E13 . 5 value and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus E13 . 5 ( black ) , * * p < 0 . 01 using ANOVA

B) Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5, and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus E13.5 (black), **p<0.01 using ANOVA test


C ) Sorted mouse thyroid cells , Tubb1 expression in endothelial cells ( Pecam - positive cells ) , epithelial cells ( Epcam ) , fibroblasts ( Pdgfra ) , and leucocytes ( CD45 ) at E17 . 5 and adulthood : quantitative PCR results ( normalized to Epcam - positive cells at adult stage and peptidylprolyl isomerase A ) . Mean ± SEM of three independent cell - sorting experiments . Statistical comparisons versus epithelial cells ( in black ) at each stage , * p < 0 . 05 using ANOVA

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C) Sorted mouse thyroid cells, Tubb1 expression in endothelial cells (Pecam-positive cells), epithelial cells (Epcam), fibroblasts (Pdgfra), and leucocytes (CD45) at E17.5 and adulthood: quantitative PCR results (normalized to Epcam-positive cells at adult stage and peptidylprolyl isomerase A). Mean±SEM of three independent cell-sorting experiments. Statistical comparisons versus epithelial cells (in black) at each stage, *p<0.05 using ANOVA test


D ) Immunohistofluorescence of β1 - tubulin ( in red ) , thyroglobulin ( TG , in green ) , and both merged ( from left to right ) at 12 GW in human thyroid tissue . The boxes delineate the enlarged areas shown

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D) Immunohistofluorescence of β1-tubulin (in red), thyroglobulin (TG, in green), and both merged (from left to right) at 12 GW in human thyroid tissue. The boxes delineate the enlarged areas shown below


F ) Localization of transfected Myc - tagged wild - type and mutant ( P160L ) β1 - tubulin in Nthy cells . Immunostaining with anti - Myc antibody ( in green ) and microtubule cytoskeleton stained with α - tubulin antibody ( in red ) . Note that β1 - tubulin - P160L is found throughout the cytoplasm showing puncta appearance , whereas wild - type β1 - tubulin colocalizes with the

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F) Localization of transfected Myc-tagged wild-type and mutant (P160L) β1-tubulin in Nthy cells. Immunostaining with anti-Myc antibody (in green) and microtubule cytoskeleton stained with α-tubulin antibody (in red). Note that β1-tubulin -P160L is found throughout the cytoplasm showing puncta appearance, whereas wild-type β1-tubulin colocalizes with the microtubules


A ) Thyroid morphology was investigated using Nkx2 - 1 staining at E9 . 5 and E11 . 5 ( sagittal sections ) and at E13 . 5 , E15 . 5 , and E17 . 5 ( transverse sections ) in wild - type ( wt ) and Tubb1 - / - littermates . Nkx2 - 1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes . Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11 . 5 and E13 . 5 ( arrow ) , respectively , in Tubb1 - / - mice . At E11 . 5 , stained migrating thyroid cells were visible along the tract only in Tubb1 - / - littermates ( arrow ) . At E17 . 5 , a pyramidal lobe was visible in Tubb1 - / - littermates ( arrow ) . tr : trachea ; ub : ultimobranchial body . Scale bar : 50

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A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μ


B ) Total thyroid surface area ( μm2 ) was quantified using Nkx2 - 1 staining at each embryonic stage . Top : thyroid surface area normalised for weight of each embryo . Bottom : thyroid surface area in μm2 . Right : proliferation ratio calculated as the proportion of Nkx2 - 1 - positive cells labelled with Ki67 at E9 . 5 in wt and Tubb1 - / - thyroid anlages . ma : median anlage . Note that the thyroid ma is larger and the proliferation ratio higher at E . 9 . 5 in Tubb1 - / - mice , whereas from E13 . 5 onwards the Tubb1 - / - thyroids were hypoplastic . Wt in black , KO , Tubb1 - / - in

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B) Total thyroid surface area (μm2) was quantified using Nkx2-1 staining at each embryonic stage. Top: thyroid surface area normalised for weight of each embryo. Bottom: thyroid surface area in μm2. Right: proliferation ratio calculated as the proportion of Nkx2-1-positive cells labelled with Ki67 at E9.5 in wt and Tubb1-/- thyroid anlages. ma: median anlage. Note that the thyroid ma is larger and the proliferation ratio higher at E.9.5 in Tubb1-/- mice, whereas from E13.5 onwards the Tubb1-/- thyroids were hypoplastic. Wt in black, KO, Tubb1-/- in gre


C ) Thyroid marker expression by quantitative PCR in thyroid tissue at E15 . 5 and E17 . 5 in Tubb1 - / - versus wt mice normalised for peptidylprolyl isomerase A . Note the decreases in thyroid differentiation markers at E15 . 5 and E17 . 5 and also in thyroid transcription factors at E17 .

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C) Thyroid marker expression by quantitative PCR in thyroid tissue at E15.5 and E17.5 in Tubb1-/- versus wt mice normalised for peptidylprolyl isomerase A. Note the decreases in thyroid differentiation markers at E15.5 and E17.5 and also in thyroid transcription factors at E17.5


D ) Staining of Nkx2 - 1 and T4 at E17 . 5 and surface area quantification demonstrating T4 retention in Tubb1 - / - versus wt thyroids . The data are the percentage of T4 or calcitonin ( CT ) surface area versus total thyroid area ( estimated from the Nkx2 - 1 - stained surface area ) . Scale bar : 50 μm . Student " s test , * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . Experiments were done on at least three tissues per stage for each

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D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen


A ) Serum TSH and T4 levels in 3 - month - old Tubb1 - / - and wild - type mice . Numbers of animals tested were 10 wild - type males and 14 Tubb1 - / - males for TSH and for 14 wild - type and 22 Tubb1 - / - males for T4 . Tubb1 - / - mice had hypothyroidism with elevated TSH and decreased T4 versus wild - type mice . Student " s test , * * p <

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A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student"s test, ** p<


B ) Nkx2 - 1 ( in brown ) immunostaining of adult thyroid tissue . Note the disorganisation of the thyroid tissue in Tubb1 - / - versus wild - type

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B) Nkx2-1 (in brown) immunostaining of adult thyroid tissue. Note the disorganisation of the thyroid tissue in Tubb1-/- versus wild-type mice


C ) Ultrastructural alterations shown by electron microscopy in wild - type and Tubb1 - / - thyroid tissues . In Tubb1 - / - tissues , note the disorganisation of secretion vesicles ( white asterisks ) and rods of identical density to secretion vesicles ( white arrow ) . The ER is considerably dilated in Tubb1 - / - thyrocytes . Representative views . Scale bars at the bottom left for each view . Co :

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C) Ultrastructural alterations shown by electron microscopy in wild-type and Tubb1-/- thyroid tissues. In Tubb1-/- tissues, note the disorganisation of secretion vesicles (white asterisks) and rods of identical density to secretion vesicles (white arrow). The ER is considerably dilated in Tubb1-/- thyrocytes. Representative views. Scale bars at the bottom left for each view. Co: colloi


D ) ER stress in Tubb1 - / - thyroid tissue . Top : Chop and XBPs expression by quantitative PCR in adults , normalized to peptidylprolyl isomerase A . Experiments with at least three tissues per stage for each genotype ; wt in black and Tubb1 - / - in grey . Bottom : Chop and Actin protein expression by western blotting in three representative wild - type and Tubb1 - / - mice . The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1 - / - versus wild - type thyroids . All lanes are from the same blot , which was cut where indicated . Student " s test , * p < 0 . 05 and * * * p <

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D) ER stress in Tubb1-/- thyroid tissue. Top: Chop and XBPs expression by quantitative PCR in adults, normalized to peptidylprolyl isomerase A. Experiments with at least three tissues per stage for each genotype; wt in black and Tubb1-/- in grey. Bottom: Chop and Actin protein expression by western blotting in three representative wild-type and Tubb1-/- mice. The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1-/- versus wild-type thyroids. All lanes are from the same blot, which was cut where indicated. Student"s test, *p<0.05 and ***p<


E ) Co - immunostaining of endoplasmic reticulum ( ER ) marker ( KDEL , in red ) , thyroglobulin ( Tg in green ) , and both merged ( from left to right ) in adult thyroid tissue . Note the thyrocyte disorganisation in Tubb1 - / -

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E) Co-immunostaining of endoplasmic reticulum (ER) marker (KDEL, in red), thyroglobulin (Tg in green), and both merged (from left to right) in adult thyroid tissue. Note the thyrocyte disorganisation in Tubb1-/- mice


A ) Representative blood smears of patients with TUBB1 mutations ( P1 and P3 homozygous for p . P160L , P4 heterozygous for p . Y106X , and P6 and P7 heterozygous for c . 35delG ) after May - Grünwald Giemsa staining . Variable platelet size in patients was observed with all three mutations with presence of macroplatelets ( arrows )

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A) Representative blood smears of patients with TUBB1 mutations (P1 and P3 homozygous for p.P160L, P4 heterozygous for p.Y106X, and P6 and P7 heterozygous for c.35delG) after May-Grünwald Giemsa staining. Variable platelet size in patients was observed with all three mutations with presence of macroplatelets (arrows)


B ) Transmission electron microscopy ( TEM ) analysis of platelet ultrastructure in the patients . The results confirm the abnormal platelet

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B) Transmission electron microscopy (TEM) analysis of platelet ultrastructure in the patients. The results confirm the abnormal platelet morphology


C ) Representative images of proplatelet formation in P1 and P4 . Cultured control and patient megakaryocytes ( MKs ) after thrombopoietin - induced differentiation were spread over a BSA - coated coverslip on day 10 . On day 13 or 14 , MKs were fixed ( 4 % paraformaldehyde ) and proplatelet structure was observed after β - tubulin staining . Scale bars : 20 μm . Only P1 and P4 could be studied . The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions ( left ) and the diameter of coiled elements ( right ) of the future proplatelets , both of which were significantly increased in the patients , indicating that the TUBB1 mutations ( homozygous p . P160L and heterozygous p . Y106X ) affected proplatelet morphology . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * * p < 0 . 001 ; 20 - 60 MKs were analysed /

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C) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat


A ) Relative expression of α - tubulin and β1 - tubulin in platelets from P1 and P3 ( homozygous p . P160L ) , P4 ( heterozygous p . Y106X ) , and P6 and P7 ( heterozygous c . 35delG ) was quantified by Western blotting using specific antibodies against α - tubulin or β1 - tubulin . With both mutations , α - tubulin expression was normal but β1 - tubulin expression was significantly decreased . The β1 - tubulin antibody recognizes C - terminal

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A) Relative expression of α-tubulin and β1-tubulin in platelets from P1 and P3 (homozygous p.P160L), P4 (heterozygous p.Y106X), and P6 and P7 (heterozygous c.35delG) was quantified by Western blotting using specific antibodies against α-tubulin or β1-tubulin. With both mutations, α-tubulin expression was normal but β1-tubulin expression was significantly decreased. The β1-tubulin antibody recognizes C-terminal domain


B ) αIIbβ3 integrin activation was evaluated by flow cytometry ( % of positive platelets and mean fluorescence intensity ) in whole blood from P1 and P3 ( homozygous p . P160L ) and from P6 and P7 ( heterozygous c . 35delG ) ; P4 could not be studied . While no αIIbβ3 integrin activations were detected in P6 and P7 ( family 3 ) , P1 and P3 ( family 1 ) exhibited a significant increase of αIIbβ3 activation , indicating basal platelet

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B) αIIbβ3 integrin activation was evaluated by flow cytometry (% of positive platelets and mean fluorescence intensity) in whole blood from P1 and P3 (homozygous p.P160L) and from P6 and P7 (heterozygous c.35delG); P4 could not be studied. While no αIIbβ3 integrin activations were detected in P6 and P7 (family 3), P1 and P3 (family 1) exhibited a significant increase of αIIbβ3 activation, indicating basal platelet activation


C ) Aggregation of washed platelets induced by ADP ( 5 or 10 μM ) or collagen ( 0 . 6 , 0 . 8 or 2 μg / mL ) was evaluated in at least one member of each family . Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls , suggesting that TUBB1 affected by homozygous p . P160L and heterozygous p . Y106X mutations affected platelet function . Platelet aggregation to both agonists was normal in both patients from family 3 ( heterozygous c . 35delG ) . Family F1 , n = 3 independent experiments ; Family F2 , n = 1 experiment ; Family F3 , n = 2 independent experiments . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , nd : statistical significance not determined

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C) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because


( C - C ' ' ' ) Terminal cell expressing reporters for membrane ( C ) , ER ( C ' ) and Golgi apparatus ( C ' ' ) . Red arrowheads : CD4 : : mIFP vesicles and associated markers at the tip of the cell .

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(C-C''') Terminal cell expressing reporters for membrane (C), ER (C') and Golgi apparatus (C''). Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.


( D ) Mean and 95 % confidence interval of tube and branch length during the first hour of growth . Numbers represent the rate of growth estimated from the slope of the curve + / - SD , p = 0 . 39 , Mann - Whitney test , n = 6 .

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(D) Mean and 95% confidence interval of tube and branch length during the first hour of growth. Numbers represent the rate of growth estimated from the slope of the curve +/-SD, p=0.39, Mann-Whitney test, n=6.


( E ) Distance of the tip of the tube , and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell . Line and shading represent mean and 95 % confidence interval , n = 4 .

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(E) Distance of the tip of the tube, and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell. Line and shading represent mean and 95% confidence interval, n=4.


( F - I ) Terminal cells expressing the general membrane marker CD4 : : mIFP in combination with different membrane or polarity markers : PH : : mCherry , a PIP2 sensor commonly used as apical plasma membrane marker ( F ' ) , an endogenously GFP - tagged Crb ( G ' ) , polarity proteins Par3 : : YFP ( H ' ) and Par6 : : mCherry ( H ' ' ) . The boxed region in ( H ' ' ' ) is shown at higher magnification in ( I ) over 4 time points . Red arrowheads : CD4 : : mIFP vesicles and associated markers at the tip of the cell .

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(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.


TEM tomograms from high - pressure frozen embryos . ( A - A ' ) Low magnification ( 2 . 5 nm voxels ) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell . ( A ' ' - A ' ' ' ) 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell .

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TEM tomograms from high-pressure frozen embryos. (A-A') Low magnification (2.5 nm voxels) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell. (A''-A''') 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell.


( B ) Example of an individual high - resolution ( 0 . 78nm voxels ) tomograms used to generate 3D reconstructions .

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(B) Example of an individual high-resolution (0.78nm voxels) tomograms used to generate 3D reconstructions.


( C ) Low resolution tomogram of another cell ; the dotted box is magnified in ( D ) . ( D ) High - resolution tomogram . ( D ' ) Model derived from 4 serial 3D reconstructions including the one shown in ( D ) .

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(C) Low resolution tomogram of another cell; the dotted box is magnified in (D). (D) High-resolution tomogram. (D') Model derived from 4 serial 3D reconstructions including the one shown in (D).


( B ) Distribution of three commonly seen classes of vesicles , expressed as their density per area of cytoplasm screened , in the two regions of the cell . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . n = 62 tomograms from two different cells . ( C ) Representative examples for each vesicle class are shown next to the quantification plots .

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(B) Distribution of three commonly seen classes of vesicles, expressed as their density per area of cytoplasm screened, in the two regions of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=62 tomograms from two different cells. (C) Representative examples for each vesicle class are shown next to the quantification plots.


( D ) Distribution of endocytic events at the apical or basal membrane compartment , expressed as density per membrane surface screened . Left shows tomograms corresponding to the cell body and branch , and right shows tomograms corresponding to the tip of the cell . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . n = 48 tomograms from two different cells .

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(D) Distribution of endocytic events at the apical or basal membrane compartment, expressed as density per membrane surface screened. Left shows tomograms corresponding to the cell body and branch, and right shows tomograms corresponding to the tip of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=48 tomograms from two different cells.


Distribution of the plasma membrane reporter PH : : GFP in control cells ( A - A ' ' ) and in cells where dynamin activity had been blocked using a temperature - sensitive allele of dynamin ( shibirets In ( B ) , was inactivated at the onset of tube formation ( C - D ) Distribution of PH : : GFP fluorescence intensity in control and shibirets cells . Data from 1 - 2 minute - interval time lapses was collected in windows of 20 minutes each ( except for t = 0 ) . ( C ) Proportion of signal in the apical and in the basal membrane compartment over time in control cells ( n = 4 ) and also in cells where dynamin was inactivated ( n = 5 ) , * p = 0 . 015 , Mann - Whitney test . ( D ) Total fluorescence intensity of PH : : GFP over time in controls ( n = 4 ) and in cells where dynamin was inactivated ( n = 5 ) , n . s . : p = 0 . 063 , Mann - Whitney test .

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Distribution of the plasma membrane reporter PH::GFP in control cells (A-A'') and in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets In (B), was inactivated at the onset of tube formation (C-D) Distribution of PH::GFP fluorescence intensity in control and shibirets cells. Data from 1-2 minute-interval time lapses was collected in windows of 20 minutes each (except for t=0). (C) Proportion of signal in the apical and in the basal membrane compartment over time in control cells (n=4) and also in cells where dynamin was inactivated (n=5), *p=0.015, Mann-Whitney test. (D) Total fluorescence intensity of PH::GFP over time in controls (n=4) and in cells where dynamin was inactivated (n=5), n.s.: p=0.063, Mann-Whitney test.


Distribution of the plasma membrane reporter PH : : GFP in cells where dynamin activity had been blocked using a temperature - sensitive allele of dynamin ( shibirets in ( E ) it was inactivated ~ 15 minutes after the tube had begun to form .

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Distribution of the plasma membrane reporter PH::GFP in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets in (E) it was inactivated ~15 minutes after the tube had begun to form.


( F - G ) Distribution of the luminal reporter Gasp : : GFP in control cells ( F ) and in cells where dynamin was inactivated ( G ) . Arrowheads point to protrusions sprouting from the lumen of the terminal cell .

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(F-G) Distribution of the luminal reporter Gasp::GFP in control cells (F) and in cells where dynamin was inactivated (G). Arrowheads point to protrusions sprouting from the lumen of the terminal cell.


( H - I ) Distribution of the general plasma membrane marker CD4 : : mIFP in combination with the Clathrin light chain fused to GFP ( CLC : : GFP , H ) and with Dynamin : : YFP ( I ) .

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(H-I) Distribution of the general plasma membrane marker CD4::mIFP in combination with the Clathrin light chain fused to GFP (CLC::GFP, H) and with Dynamin::YFP (I).


( A - F ) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos . ( A ) Control . ( B - F ) Kept at 34ºC for 1 ( B , E ) or for 2 hours ( C , D , F ) . Boxed regions in ( A ) and ( B ) are magnified in ( A ' ' ) and ( B ' ' ) . ( A ' , B ' ) Reconstructed 3D models . The tube membrane ( cyan ) is characterized by the electron - dense apical extracellular matrix ( aECM ) . Invaginations with connections that can be traced to the tube membrane are red , invaginations from the basal plasma membrane are green . The basal membrane is light pink . ( C - D ' ) shibirets terminal cell after 2 hours at 34ºC . ( C ' ) 3D reconstruction , color coding for basal and tube membrane is the same as above . Membrane that is continuous with both the apical and the basal membrane is shown in green , purple and dark blue . ( D - D ' ) higher magnification of a region in the same cell at a level where membrane sheets ( purple , dark blue ) bridge the apical and basal plasma membrane domains . ( E - E ' ' ) Two focal planes and model of a tomogram from the cell shown in ( B ) where a basal ( pink arrowhead ) and an apical invagination ( blue ) are seen in close proximity ( closest distance is marked by cyan and blue arrowheads ) . ( F - F ' ) apical membrane overgrowth regions of the cell shown in ( C ) . The genotype of the embryos was shibirets ; btl > KDEL : : RFP , Par3 : : YFP . The cell shown in ( C - D ) was found and acquired without the CLEM approach .

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(A-F) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos. (A) Control. (B-F) Kept at 34ºC for 1 (B, E) or for 2 hours (C, D, F). Boxed regions in (A) and (B) are magnified in (A'') and (B''). (A', B') Reconstructed 3D models. The tube membrane (cyan) is characterized by the electron-dense apical extracellular matrix (aECM). Invaginations with connections that can be traced to the tube membrane are red, invaginations from the basal plasma membrane are green. The basal membrane is light pink. (C-D') shibirets terminal cell after 2 hours at 34ºC. (C') 3D reconstruction, color coding for basal and tube membrane is the same as above. Membrane that is continuous with both the apical and the basal membrane is shown in green, purple and dark blue. (D-D') higher magnification of a region in the same cell at a level where membrane sheets (purple, dark blue) bridge the apical and basal plasma membrane domains. (E-E'') Two focal planes and model of a tomogram from the cell shown in (B) where a basal (pink arrowhead) and an apical invagination (blue) are seen in close proximity (closest distance is marked by cyan and blue arrowheads). (F-F') apical membrane overgrowth regions of the cell shown in (C). The genotype of the embryos was shibirets; btl>KDEL::RFP, Par3::YFP. The cell shown in (C-D) was found and acquired without the CLEM approach.


( A - B ) Terminal cells expressing the membrane marker CD4 : : mIFP under btl - gal4 and FGFR : : GFP under its own promoter ( from the fTRG library ) . ( A ) Time lapse imaging of a control cell . Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4 : : mIFP and FGFR : : GFP . ( B ) shibirets cell imaged before dynamin inactivation , after 23 and 45 minutes of inactivation , and after 30 minutes of recovery . White arrowheads point to FGFR : : GFP accumulation at the apical compartment .

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(A-B) Terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. White arrowheads point to FGFR::GFP accumulation at the apical compartment.


( C - E ) Single confocal planes of terminal cells expressing PH : : mCherry and stained for βPS - Integrin . The outline of the cells was traced using the PH : : mCherry signal and is shown as a blue dashed line . ( C ) Control . Blue arrowheads : βPS - Integrin signal in filopodia . ( D ) 1 hour at 34ºC , ( E ) 2 hours at 34ºC . ( C ' ' - E ' ' ) Orthogonal views of the lines shown in white in ( C - E ) .

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(C-E) Single confocal planes of terminal cells expressing PH::mCherry and stained for βPS-Integrin. The outline of the cells was traced using the PH::mCherry signal and is shown as a blue dashed line. (C) Control. Blue arrowheads: βPS-Integrin signal in filopodia. (D) 1 hour at 34ºC, (E) 2 hours at 34ºC. (C''-E'') Orthogonal views of the lines shown in white in (C-E).


( F - G ) Quantification of fluorescence intensity from stained embryos of FGFR : : GFP ( F ) and of βPS - Integrin ( G ) . Data are plotted as mean + / - SD ; significance was assessed using one - way ANOVA with Dunnett ' s correction for multiple comparisons . * * p = 0 . 0022 , * * * p = 0 . 0003 , * * * * p < 0 . 0001 . Number of cells analyzed , for ( F ) : Control n = 8 , 1hr at 34ºC n = 6 , Recovery n = 8 . ( G ) . For ( G ) : Control = 7 , 1hr at 34ºC = 8 , 2hrs at 34ºC = 8 .

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(F-G) Quantification of fluorescence intensity from stained embryos of FGFR::GFP (F) and of βPS-Integrin (G). Data are plotted as mean +/- SD; significance was assessed using one-way ANOVA with Dunnett's correction for multiple comparisons. **p=0.0022, ***p=0.0003, ****p<0.0001. Number of cells analyzed, for (F): Control n=8, 1hr at 34ºC n=6, Recovery n=8. (G). For (G): Control= 7, 1hr at 34ºC= 8, 2hrs at 34ºC= 8.


High time resolution imaging of the membrane marker CD4 : : mIFP expressed under btl - gal4 . ( A ) Example of a large CD4 vesicle ( arrowhead ) rapidly moving towards the tip of the terminal cell . ( B - D ) Analysis of 28 CD4 vesicles in five terminal cells .

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High time resolution imaging of the membrane marker CD4::mIFP expressed under btl-gal4. (A) Example of a large CD4 vesicle (arrowhead) rapidly moving towards the tip of the terminal cell. (B-D) Analysis of 28 CD4 vesicles in five terminal cells.


( B ) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot . ( C ) Rose diagram of the trajectories shown in ( B ) . ( D ) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min . Data are plotted as mean + / - SD .

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(B) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot. (C) Rose diagram of the trajectories shown in (B). (D) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min. Data are plotted as mean +/-SD.


( E - H ) Terminal cells expressing the membrane reporter CD4 : : mIFP together with markers for vesicles of the endocytic pathway . ( E ) Rab5 : : YFP , red arrowheads point to a Rab5 - positive vesicle ; the white arrowhead shows the same vesicle once it lost the Rab5 signal . ( F ) FYVE : : GFP , a PI3P reporter and Spin : : mRFP , a lysosomal marker . Black arrowheads point to small FYVE : : GFP vesicles and the squared area shows large ones at the tip . ( G ) Par6 : : mCherry and endogenously labelled Rab7 : : YFP . ( H ) Percentage of CD4 : : mIFP vesicles that carry the indicated markers . Data are plotted as mean + / - SD . For each marker we analyzed at least 20 minutes of cell growth with 10 - 20 timepoints . Number of cells analyzed : Par3 , n = 2 ; Rab5 , n = 3 ; FYVE : : GFP , n = 4 ; FYVE : : GFP - Spin : : RFP ( which includes single - and double - positive vesicles ) , n = 3 ; dLamp1 , n = 2 ; Rab7 , n = 5 .

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(E-H) Terminal cells expressing the membrane reporter CD4::mIFP together with markers for vesicles of the endocytic pathway. (E) Rab5::YFP, red arrowheads point to a Rab5-positive vesicle; the white arrowhead shows the same vesicle once it lost the Rab5 signal. (F) FYVE::GFP, a PI3P reporter and Spin::mRFP , a lysosomal marker. Black arrowheads point to small FYVE::GFP vesicles and the squared area shows large ones at the tip. (G) Par6::mCherry and endogenously labelled Rab7::YFP. (H) Percentage of CD4::mIFP vesicles that carry the indicated markers. Data are plotted as mean +/-SD. For each marker we analyzed at least 20 minutes of cell growth with 10-20 timepoints. Number of cells analyzed: Par3, n=2; Rab5, n=3; FYVE::GFP, n=4; FYVE::GFP-Spin::RFP (which includes single- and double-positive vesicles), n=3; dLamp1, n=2; Rab7, n=5.


( A - F ) Terminal cells expressing CD4 : : mIFP and FYVE : : GFP under btl - gal4 . The outline of the cells was traced using the CD4 : : mIFP signal and is shown as a blue dashed line . ( A - C ) Control cell . ( D - F ) shibirets cell before 34ºC ( D ) , after 30 minutes at 34ºC ( E ) and after 30 minutes of recovery ( F ) . Red arrowheads : FYVE : : GFP puncta .

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(A-F) Terminal cells expressing CD4::mIFP and FYVE::GFP under btl-gal4. The outline of the cells was traced using the CD4::mIFP signal and is shown as a blue dashed line. (A-C) Control cell. (D-F) shibirets cell before 34ºC (D), after 30 minutes at 34ºC (E) and after 30 minutes of recovery (F). Red arrowheads: FYVE::GFP puncta.


( G - H ) Number of FYVE : : GFP vesicles in 4 control cells ( G ) and in 6 shibirets cells ( H ) . Box plots represent median , interquartile range ( IQR ) , and IQR * 1 . 5 below and above the IQR . Significance in ( H ) was assessed using one - way ANOVA with Geisser - Greenhouse correction for paired data and Dunnett ' s multiple comparisons test . * p = 0 . 0288 , * * * p = 0 . 0016 .

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(G-H) Number of FYVE::GFP vesicles in 4 control cells (G) and in 6 shibirets cells (H). Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. Significance in (H) was assessed using one-way ANOVA with Geisser-Greenhouse correction for paired data and Dunnett's multiple comparisons test. *p=0.0288, ***p=0.0016.


( I ) shrbO3 / G5 mutant cell expressing CD4 : : mIFP under btl - gal4 . Arrowhead : CD4 : : mIFP aggregation .

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(I) shrbO3/G5 mutant cell expressing CD4::mIFP under btl-gal4. Arrowhead: CD4::mIFP aggregation.


( J - K ) Terminal cells over - expressing Shrb : : GFP under btl - gal4 together with PH : : mCherry ( J ) , or together with Spin : : RFP and CD4 : : mIFP ( K ) . Arrowheads : Shrb : : GFP accumulations .

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(J-K) Terminal cells over-expressing Shrb::GFP under btl-gal4 together with PH::mCherry (J), or together with Spin::RFP and CD4::mIFP (K). Arrowheads: Shrb::GFP accumulations.


( L - M ) Dorsal trunk cells stained for Serp . In control embryos ( L ) , Serp is seen exclusively in the tracheal lumen . The cells themselves are not visible in these images . In embryos expressing Shrb : : GFP under btl - gal4 ( M ) , Serp is also seen inside the cells , usually in association with or surrounded by Shrb : : GFP . Boxed regions are magnified in ( L ' - M ' ) and shown as single confocal planes .

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(L-M) Dorsal trunk cells stained for Serp. In control embryos (L), Serp is seen exclusively in the tracheal lumen. The cells themselves are not visible in these images. In embryos expressing Shrb::GFP under btl-gal4 (M), Serp is also seen inside the cells, usually in association with or surrounded by Shrb::GFP. Boxed regions are magnified in (L'-M') and shown as single confocal planes.


A , Representative images of DIV 3 + 2 cortical neurons transfected with FlucSM - EGFP ( green ) and subjected to the indicated treatments : 5 µM MG - 132 for 4 hours ; 10 nM Bafilomycin A1 ( BafA1 ) for 24 hours ; 0 . 5 µM 17 - AAG for 4 hours ; heat shock at 43 ° C for 30 min . Nuclei were labeled with DAPI ( blue ) . Insets show higher magnification of the areas outlined by the boxes .

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A, Representative images of DIV 3+2 cortical neurons transfected with FlucSM-EGFP (green) and subjected to the indicated treatments: 5 µM MG-132 for 4 hours; 10 nM Bafilomycin A1 (BafA1) for 24 hours; 0.5 µM 17-AAG for 4 hours; heat shock at 43°C for 30 min. Nuclei were labeled with DAPI (blue). Insets show higher magnification of the areas outlined by the boxes.


D , DIV 3 + 2 cortical neurons co - transfected with FlucWT - EGFP ( green ) and HTT - Q97 - mCherry ( magenta ) and treated with 1 mM 4 - PBA ( lower row ) or vehicle control ( upper row ) from DIV 3 . Cells were stained for MAP2 ( grey ) as a neuronal marker , nuclei were labeled with DAPI ( blue ) . Arrowhead points to Fluc - EGFP foci . Corresponding cultures transfected with control HTT are shown in Fig . EV1D . E , Quantification of the fraction of double - transfected cells showing Fluc - EGFP foci . N = 4 biological replicates . Two - way ANOVA with Tukey ' s multiple comparisons test . ANOVA : HTT , * * p = 0 . 0046 ; 4 - PBA , n . s . ; HTT x 4 - PBA , n . s . Significant pairwise comparisons are indicated on the graph .

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D, DIV 3+2 cortical neurons co-transfected with FlucWT-EGFP (green) and HTT-Q97-mCherry (magenta) and treated with 1 mM 4-PBA (lower row) or vehicle control (upper row) from DIV 3. Cells were stained for MAP2 (grey) as a neuronal marker, nuclei were labeled with DAPI (blue). Arrowhead points to Fluc-EGFP foci. Corresponding cultures transfected with control HTT are shown in Fig. EV1D. E, Quantification of the fraction of double-transfected cells showing Fluc-EGFP foci. N=4 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test. ANOVA: HTT, **p=0.0046; 4-PBA, n.s.; HTT x 4-PBA, n.s. Significant pairwise comparisons are indicated on the graph. D


C , Left , Western blot of acute brain slice lysates from Fluc - EGFP mice and non - transgenic littermates . The slices were treated with 1 mM 4 - PBA or vehicle control , and subjected to heat shock at 43 ° C for 15 min , as indicated above the blot . Middle , quantification of specific luciferase activity of Fluc - EGFP upon indicated treatments , normalized to vehicle - treated slices kept at 37 ° C . Right , quantification of Fluc - EGFP protein levels in the indicated conditions . N = 3 mice . Colored asterisks indicate comparisons to the corresponding vehicle - treated 37 ° C control group ( one - sample t - test ) , black asterisk indicates comparison between 4 - PBA and vehicle - treated heat shock groups ( two - tailed t - test ) .

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C, Left, Western blot of acute brain slice lysates from Fluc-EGFP mice and non-transgenic littermates. The slices were treated with 1 mM 4-PBA or vehicle control, and subjected to heat shock at 43°C for 15 min, as indicated above the blot. Middle, quantification of specific luciferase activity of Fluc-EGFP upon indicated treatments, normalized to vehicle-treated slices kept at 37°C. Right, quantification of Fluc-EGFP protein levels in the indicated conditions. N=3 mice. Colored asterisks indicate comparisons to the corresponding vehicle-treated 37°C control group (one-sample t-test), black asterisk indicates comparison between 4-PBA and vehicle-treated heat shock groups (two-tailed t-test).


D , Representative Western blot of hippocampal lysates from Fluc - EGFP mice at the indicated ages . Short bracket with an arrow indicates the high molecular weight species observed in older mice . Long bracket indicates the part of the lane that was used for Fluc protein quantification ( see Materials and Methods ) . Several lanes on the blot between 6 - month - old and 12 - month - old samples were digitally removed .

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D, Representative Western blot of hippocampal lysates from Fluc-EGFP mice at the indicated ages. Short bracket with an arrow indicates the high molecular weight species observed in older mice. Long bracket indicates the part of the lane that was used for Fluc protein quantification (see Materials and Methods). Several lanes on the blot between 6-month-old and 12-month-old samples were digitally removed.


A , Cortical sections from rTg4510 : Fluc - EGFP mice ( right ) and control littermates ( left ) stained for total human tau ( HT7 , magenta ) and p - tau ( AT8 , yellow ) . Fluc - EGFP was detected by EGFP fluorescence ( green ) , nuclei were labeled with DAPI ( blue ) . Images on the right show higher magnification of the areas indicated by the boxes , with neuronal cell bodies outlined . Note the presence of EGFP foci ( arrowheads ) in the majority of neurons in rTg4510 : Fluc - EGFP mice . Occasional foci of lower relative fluorescence intensity can be observed in control mice ( arrow ) .

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A, Cortical sections from rTg4510:Fluc-EGFP mice (right) and control littermates (left) stained for total human tau (HT7, magenta) and p-tau (AT8, yellow). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were labeled with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes, with neuronal cell bodies outlined. Note the presence of EGFP foci (arrowheads) in the majority of neurons in rTg4510:Fluc-EGFP mice. Occasional foci of lower relative fluorescence intensity can be observed in control mice (arrow).


D , Quantification of the fraction of cells with EGFP foci among the cells with and without tau expression and p - tau pathology . N = 4 rTg4510 : Fluc - EGFP mice , 3 WT : Fluc - EGFP mice , 3 CaMKIIα - tTA : Fluc - EGFP mice and 3 tetO - tauP301L : Fluc - EGFP mice . WT : Fluc - EGFP , CaMKIIα - tTA : Fluc - EGFP and tetO - tauP301L : Fluc - EGFP littermates did not differ from each other and were pooled together as controls . One - way ANOVA with Tukey ' s multiple comparisons test . ANOVA : * * * * p < 0 . 0001 . Pairwise comparisons between groups are indicated on the graph .

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D, Quantification of the fraction of cells with EGFP foci among the cells with and without tau expression and p-tau pathology. N=4 rTg4510:Fluc-EGFP mice, 3 WT:Fluc-EGFP mice, 3 CaMKIIα-tTA:Fluc-EGFP mice and 3 tetO-tauP301L:Fluc-EGFP mice. WT:Fluc-EGFP, CaMKIIα-tTA:Fluc-EGFP and tetO-tauP301L:Fluc-EGFP littermates did not differ from each other and were pooled together as controls. One-way ANOVA with Tukey's multiple comparisons test. ANOVA: ****p < 0.0001. Pairwise comparisons between groups are indicated on the graph.


A , Cortical ( upper row ) and striatal ( lower row ) sections from 12 - week - old R6 / 2 : Fluc - EGFP mice ( right ) and control WT : Fluc - EGFP littermates ( left ) stained for aggregated mHTT ( EM48 , magenta ) . Fluc - EGFP was detected by EGFP fluorescence ( green ) , nuclei were counterstained with DAPI ( blue ) . Images on the right show higher magnification of the areas indicated by the boxes . Continuous and stippled lines mark cell bodies and nuclei , respectively .

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A, Cortical (upper row) and striatal (lower row) sections from 12-week-old R6/2:Fluc-EGFP mice (right) and control WT:Fluc-EGFP littermates (left) stained for aggregated mHTT (EM48, magenta). Fluc-EGFP was detected by EGFP fluorescence (green), nuclei were counterstained with DAPI (blue). Images on the right show higher magnification of the areas indicated by the boxes. Continuous and stippled lines mark cell bodies and nuclei, respectively.


Luciferase assay in R6 / 2 mice . C , Fluc - EGFP protein quantity in the indicated brain regions of R6 / 2 : Fluc - EGFP mice measured by Western blot . Values are normalized to WT : Fluc - EGFP littermates . N = 5 mice of each genotype . Colored asterisks in B and C indicate significant comparisons to the respective brain regions of control littermates ( one - sample t - test ) .

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Luciferase assay in R6/2 mice. C, Fluc-EGFP protein quantity in the indicated brain regions of R6/2:Fluc-EGFP mice measured by Western blot. Values are normalized to WT:Fluc-EGFP littermates. N=5 mice of each genotype. Colored asterisks in B and C indicate significant comparisons to the respective brain regions of control littermates (one-sample t-test).


A , B , Primary cortical neurons transfected with cyt - Fluc - EGFP ( A ) or nuc - Fluc - EGFP ( B ) ( green ) , in combination with nuc - mHTT ( upper rows ) or cyt - mHTT ( lower rows ) ( magenta ) . Cells were fixed at DIV 3 + 2 and stained for the neuronal marker MAP2 ( grey ) . Nuclei were labeled with DAPI ( blue ) . Arrowheads point to Fluc - EGFP foci . Dashed lines mark the nuclei . Schemes on the right summarize the cellular distribution of the respective Fluc - EGFP ( green ) and mHTT ( magenta ) proteins . Corresponding cultures transfected with control HTT are shown in Appendix Fig . S3A .

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A, B, Primary cortical neurons transfected with cyt-Fluc-EGFP (A) or nuc-Fluc-EGFP (B) (green), in combination with nuc-mHTT (upper rows) or cyt-mHTT (lower rows) (magenta). Cells were fixed at DIV 3+2 and stained for the neuronal marker MAP2 (grey). Nuclei were labeled with DAPI (blue). Arrowheads point to Fluc-EGFP foci. Dashed lines mark the nuclei. Schemes on the right summarize the cellular distribution of the respective Fluc-EGFP (green) and mHTT (magenta) proteins. Corresponding cultures transfected with control HTT are shown in Appendix Fig. S3A.


A . Histograms of crossovers ( blue ) per F2 individual in the indicated populations , with the Poisson expectation plotted in red . Mean values are indicated by the black dotted lines . The genome average SNPs / kb for each cross are printed above the plots .

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A. Histograms of crossovers (blue) per F2 individual in the indicated populations, with the Poisson expectation plotted in red. Mean values are indicated by the black dotted lines. The genome average SNPs/kb for each cross are printed above the plots.


B . Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes . Mean values are shown by horizontal dashed lines . SNPs per 200 kb are plotted and shaded in colour ( Col × Ws ( purple ) , Col × Bur ( Lawrence et al , 2019 ) ( dark green ) , Col × Ct ( light blue ) , Col × Ler F2 populations with 17 , 077 ( red ) and 1 , 840 ( blue ) crossovers ( Serra et al , 2018b ; Rowan et al , 2019 ) and Col × CLC ( pink / light green , where pink indicates Cvi SNPs and green indicates Ler SNPs ) ) . The positions of telomeres ( TEL ) and centromeres ( CEN ) are indicated . The number of crossovers analysed is printed inset . C . Data as for B , but analyzing crossovers ( lines ) or SNPs ( shading ) along proportionally scaled chromosome arms , orientated from telomere ( TEL ) to centromere ( CEN ) .

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B. Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes. Mean values are shown by horizontal dashed lines. SNPs per 200 kb are plotted and shaded in colour (Col×Ws (purple), Col×Bur (Lawrence et al, 2019) (dark green), Col×Ct (light blue), Col×Ler F2 populations with 17,077 (red) and 1,840 (blue) crossovers (Serra et al, 2018b; Rowan et al, 2019) and Col×CLC (pink/light green, where pink indicates Cvi SNPs and green indicates Ler SNPs)). The positions of telomeres (TEL) and centromeres (CEN) are indicated. The number of crossovers analysed is printed inset. C. Data as for B, but analyzing crossovers (lines) or SNPs (shading) along proportionally scaled chromosome arms, orientated from telomere (TEL) to centromere (CEN).


A . Crossover frequency normalised by the number of F2 individuals , and SNP density in 100 kilobase ( kb ) adjacent windows were calculated for each population and ranked into percentiles according to SNP density . Centromeric regions were excluded from analysis ( Underwood et al , 2018 ) . The Spearman ' s rank correlation coefficient ( r ) between SNP density and crossover frequency is printed inset . Trend lines were fitted in ggplot using a generalized additive model ( GAM ) with the formula y ~ poly ( x , 2 ) .

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A. Crossover frequency normalised by the number of F2 individuals, and SNP density in 100 kilobase (kb) adjacent windows were calculated for each population and ranked into percentiles according to SNP density. Centromeric regions were excluded from analysis (Underwood et al, 2018). The Spearman's rank correlation coefficient (r) between SNP density and crossover frequency is printed inset. Trend lines were fitted in ggplot using a generalized additive model (GAM) with the formula y~poly(x,2).


A . SNPs / kb in physical windows of increasing size ( kb ) around crossover midpoints ( red ) , or for matched randomly chosen positions ( grey ) . Printed above the plot for each window are circles coloured green if crossover SNPs / kb values are significantly different to random ( P < 0 . 05 ) , or red if not ( P > 0 . 05 ) ( Bonferroni adjusted t - tests ) . The population ( Col × Ws , Col × Bur ( Lawrence et al , 2019 ) , Col × Ct , Col × Ler ( Serra et al , 2018b ; Rowan et al , 2019 ) and Col × CLC ) is printed above each plot , and the number of positions analysed is printed inset .

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A. SNPs/kb in physical windows of increasing size (kb) around crossover midpoints (red), or for matched randomly chosen positions (grey). Printed above the plot for each window are circles coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population (Col×Ws, Col×Bur (Lawrence et al, 2019), Col×Ct, Col×Ler (Serra et al, 2018b; Rowan et al, 2019) and Col×CLC) is printed above each plot, and the number of positions analysed is printed inset.


B . As for A , but analyzing the indicated Col / Ler transition and transversion polymorphisms around Col × Ler crossovers ( Rowan et al , 2019 ) ( red ) , or the same number of random positions ( grey ) .

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B. As for A, but analyzing the indicated Col/Ler transition and transversion polymorphisms around Col×Ler crossovers (Rowan et al, 2019) (red), or the same number of random positions (grey).


C . As for A , but analysing Col / Ler SNPs / kb around crossovers from wild type , HEI10 - OE , recq4a recq4b or HEI10 - OE recq4a recq4b ( Serra et al , 2018b ) . All populations were generated from Col × Ler hybrids . Printed beneath are the estimated average numbers of Class I and Class II crossovers per meiosis for each genotype , measured via genotyping - by - sequencing ( Serra et al , 2018b ; Ziolkowski et al , 2017 ) . To the right are plots showing the significance of SNPs / kb differences between crossover sets , across the windows tested ( Bonferroni adjusted t - tests ) .

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C. As for A, but analysing Col/Ler SNPs/kb around crossovers from wild type, HEI10-OE, recq4a recq4b or HEI10-OE recq4a recq4b (Serra et al, 2018b). All populations were generated from Col×Ler hybrids. Printed beneath are the estimated average numbers of Class I and Class II crossovers per meiosis for each genotype, measured via genotyping-by-sequencing (Serra et al, 2018b; Ziolkowski et al, 2017). To the right are plots showing the significance of SNPs/kb differences between crossover sets, across the windows tested (Bonferroni adjusted t-tests).


A . Histograms of observed crossovers per F2 individual in the indicated wild type and msh2 populations , from Col × Ct , Col × Ler and Col × CLC crosses ( blue ) . The Poisson expectation is plotted in red . Data from HEI10 - OE , recq4a recq4b and HEI10 - OE recq4a recq4b F2 populations are also shown for comparison ( Serra et al , 2018b ; Ziolkowski et al , 2017 ) . Mean values are indicated by black dotted lines .

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A. Histograms of observed crossovers per F2 individual in the indicated wild type and msh2 populations, from Col×Ct, Col×Ler and Col×CLC crosses (blue). The Poisson expectation is plotted in red. Data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b F2 populations are also shown for comparison (Serra et al, 2018b; Ziolkowski et al, 2017). Mean values are indicated by black dotted lines.


B . Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes , with mean values shown by horizontal dashed lines . Data are shown for wild type ( red ) and msh2 ( blue ) crossovers generated from Col × Ct , Col × Ler and Col × CLC hybrids . SNPs per 200 kb are shaded in colour ( blue = Col / Ct , green = Col / Ler , pink = Col / Cvi ) . The positions of telomeres ( TEL ) and centromeres ( CEN ) are labeled . The number of crossovers analysed is printed inset . C . Data as for B , but analyzing crossovers in wild type ( red ) and msh2 ( blue ) , or SNPs along proportionally scaled chromosome arms orientated from telomeres ( TEL ) to centromeres ( CEN ) . Crossover data from HEI10 - OE , recq4a recq4b and HEI10 - OE recq4a recq4b Col × Ler F2 populations are also shown ( Serra et al , 2018b ; Ziolkowski et al , 2017 ) .

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B. Crossovers per 200 kb per F2 plotted along the Arabidopsis chromosomes, with mean values shown by horizontal dashed lines. Data are shown for wild type (red) and msh2 (blue) crossovers generated from Col×Ct, Col×Ler and Col×CLC hybrids. SNPs per 200 kb are shaded in colour (blue=Col/Ct, green=Col/Ler, pink=Col/Cvi). The positions of telomeres (TEL) and centromeres (CEN) are labeled. The number of crossovers analysed is printed inset. C. Data as for B, but analyzing crossovers in wild type (red) and msh2 (blue), or SNPs along proportionally scaled chromosome arms orientated from telomeres (TEL) to centromeres (CEN). Crossover data from HEI10-OE, recq4a recq4b and HEI10-OE recq4a recq4b Col×Ler F2 populations are also shown (Serra et al, 2018b; Ziolkowski et al, 2017).


A . Histograms showing the mean distance ( megabases , Mb ) of observed double crossovers ( DCO , red vertical line ) , compared to 2 , 000 matched sets of randomly generated distances ( RAN , black ) . The mean distance of the random sets is shown in by the vertical grey line .

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A. Histograms showing the mean distance (megabases, Mb) of observed double crossovers (DCO, red vertical line), compared to 2,000 matched sets of randomly generated distances (RAN, black). The mean distance of the random sets is shown in by the vertical grey line.


B . SNPs / kb values were calculated in physical windows of increasing size ( kb ) around crossover midpoints in wild type ( red ) and msh2 ( blue ) . The same values were calculated for matched randomly chosen positions ( grey ) . Printed above the plot for each window are circles that are coloured green if crossover SNPs / kb values are significantly different to random ( P < 0 . 05 ) , or red if not ( P > 0 . 05 ) ( Bonferroni adjusted t - tests ) . The population analysed ( Col × Ct , Col × Ler , Col × CLC ) and its genome average SNP frequency ( SNPs / kb ) is printed above the plots and the number of crossover positions analysed is printed inset . Beneath are plots showing the significance of SNPs / kb differences around crossovers between the indicated genotypes .

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B. SNPs/kb values were calculated in physical windows of increasing size (kb) around crossover midpoints in wild type (red) and msh2 (blue). The same values were calculated for matched randomly chosen positions (grey). Printed above the plot for each window are circles that are coloured green if crossover SNPs/kb values are significantly different to random (P<0.05), or red if not (P>0.05) (Bonferroni adjusted t-tests). The population analysed (Col×Ct, Col×Ler, Col×CLC) and its genome average SNP frequency (SNPs/kb) is printed above the plots and the number of crossover positions analysed is printed inset. Beneath are plots showing the significance of SNPs/kb differences around crossovers between the indicated genotypes.


A . Representative DAPI - stained spreads of pachytene , diakinesis , metaphase - I , dyad and tetrad meiotic stages , for wild type and msh2 , in Col × Ler or Col × CLC hybrid backgrounds .

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A. Representative DAPI-stained spreads of pachytene, diakinesis, metaphase-I, dyad and tetrad meiotic stages, for wild type and msh2, in Col×Ler or Col×CLC hybrid backgrounds.


B . Male meiocytes immunostained for MSH2 ( red ) and ASY1 ( green ) , and stained for DAPI ( blue ) in wild type ( Col ) or msh2 .

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B. Male meiocytes immunostained for MSH2 (red) and ASY1 (green), and stained for DAPI (blue) in wild type (Col) or msh2.


C . As for B , but immunostaining wild type male meiocytes for MSH2 ( red ) , MSH4 ( green ) and staining chromatin with DAPI ( blue ) .

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C. As for B, but immunostaining wild type male meiocytes for MSH2 (red), MSH4 (green) and staining chromatin with DAPI (blue).


A . Crossover frequency ( black ) and SNP density ( purple ) per 200 kb measured from the Col × Ct F2 population . The position of the centromere ( CEN , vertical dashed line ) and 420 FTL transgenes ( red and green vertical lines ) used to measure crossover frequency are indicated .

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A. Crossover frequency (black) and SNP density (purple) per 200 kb measured from the Col×Ct F2 population. The position of the centromere (CEN, vertical dashed line) and 420 FTL transgenes (red and green vertical lines) used to measure crossover frequency are indicated.


B . Plots of chromosome 3 showing positions of Col / Col ( black ) or Col / Ct ( purple ) homozygous or heterozygous genotypes at the indicated marker positions ( circles ) . The positions of the 420 FTL transgenes are indicated ( red and green vertical lines ) .

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B. Plots of chromosome 3 showing positions of Col/Col (black) or Col/Ct (purple) homozygous or heterozygous genotypes at the indicated marker positions (circles). The positions of the 420 FTL transgenes are indicated (red and green vertical lines).


C . 420 crossover frequency ( cM ) in the HOM - HOM , HET - HET , HET - HOM , HOM - HET genotypes shown in B , in either wild type or msh2 . Values for replicate individuals are plotted , in addition to the mean ( red ) .

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C. 420 crossover frequency (cM) in the HOM-HOM, HET-HET, HET-HOM, HOM-HET genotypes shown in B, in either wild type or msh2. Values for replicate individuals are plotted, in addition to the mean (red).


D . As for C , but comparing genotypes in wild type or HEI10 - OE backgrounds . Values for replicate individuals are plotted , in addition to the mean ( red ) .

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D. As for C, but comparing genotypes in wild type or HEI10-OE backgrounds. Values for replicate individuals are plotted, in addition to the mean (red).


D . Plots of SNP density per 10 kb along the chromosomes for Arabidopsis Col × Ler and budding yeast S288c × SK1 crosses ( Cooper et al , 2018 ) . Telomere positions are shown by vertical lines , and centromeres by vertical dashed lines . Centromeres are also labeled above the plots in red , as ' CEN ' or ' C ' .

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D. Plots of SNP density per 10 kb along the chromosomes for Arabidopsis Col×Ler and budding yeast S288c×SK1 crosses (Cooper et al, 2018). Telomere positions are shown by vertical lines, and centromeres by vertical dashed lines. Centromeres are also labeled above the plots in red, as 'CEN' or 'C'.


Cell counting experiments analyzing the response to trastuzumab ( 50 nM , 5 days ) of naïve BT474 as well as the resistant BTRH pool and the BTRH # 10 clone .

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Cell counting experiments analyzing the response to trastuzumab (50 nM, 5 days) of naïve BT474 as well as the resistant BTRH pool and the BTRH#10 clone.


Western blot analyses of the expression of HER2 , pHER2 , HER3 and pHER3 in BT474 , BTRH and BTRH # 10 cells . HER2 and HER3 were immunoprecipitated from 100 or 500 μg of cell extracts , respectively , and blots probed with anti - HER2 , anti - HER3 or anti - phosphotyrosine . Calnexin was used as a loading control . Numbers below the blots show the quantitation of the signal of each band , referred to BT474 cells ( intensity of band in BT474 cells = 1 ) .

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Western blot analyses of the expression of HER2, pHER2, HER3 and pHER3 in BT474, BTRH and BTRH#10 cells. HER2 and HER3 were immunoprecipitated from 100 or 500 μg of cell extracts, respectively, and blots probed with anti-HER2, anti-HER3 or anti-phosphotyrosine. Calnexin was used as a loading control. Numbers below the blots show the quantitation of the signal of each band, referred to BT474 cells (intensity of band in BT474 cells=1).


Analyses of cell surface levels of HER2 and HER3 in BT474 and BTRH cells by surface immunoprecipitation .

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Analyses of cell surface levels of HER2 and HER3 in BT474 and BTRH cells by surface immunoprecipitation.


Response of BT474 , BTRH and BTRH # 10 to EV20 / MMAF . Cells treated with the indicated doses of EV20 / MMAF for 5 days were counted .

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Response of BT474, BTRH and BTRH#10 to EV20/MMAF. Cells treated with the indicated doses of EV20/MMAF for 5 days were counted.


Action of the nude EV20 antibody or EV20 / MMAF ( 10 nM each , 5 days ) on BT474 , BTRH and BTRH # 10 cells .

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Action of the nude EV20 antibody or EV20/MMAF (10 nM each, 5 days) on BT474, BTRH and BTRH#10 cells.


Comparison of the effect of MMAF and EV20 / MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs .

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Comparison of the effect of MMAF and EV20/MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs.


Response to trastuzumab ( 50 nM , 5 days ) of BT474 , BTRH , patient derived cells ( PDX118 ) and their trastuzumab - resistant derivatives TR1 and TR2 .

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Response to trastuzumab (50 nM, 5 days) of BT474, BTRH, patient derived cells (PDX118) and their trastuzumab-resistant derivatives TR1 and TR2.


HER2 , HER3 and pHER3 levels in BT474 , BTRH , PDX118 , TR1 and TR2 . pHER3 levels were analyzed by a specific anti - pHER3 antibody .

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HER2, HER3 and pHER3 levels in BT474, BTRH, PDX118, TR1 and TR2. pHER3 levels were analyzed by a specific anti-pHER3 antibody.


Effect of EV20 / MMAF on BT474 , BTRH , PDX118 , TR1 and TR2 . Cells were counted after 5 days of treatment with the indicated doses of EV20 / MMAF .

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Effect of EV20/MMAF on BT474, BTRH, PDX118, TR1 and TR2. Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.


Effect of trastuzumab ( 50 nM , 5 days ) in HER2 positive breast cancer cell lines ( SKBR3 , BT474 , BTRH , MDA - MB - 361 , HCC1419 , HCC1569 and HCC1954 ) . The blue line indicates the threshold ( established from BTRH ) used to consider a cell line resistant to trastuzumab .

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Effect of trastuzumab (50 nM, 5 days) in HER2 positive breast cancer cell lines (SKBR3, BT474, BTRH, MDA-MB-361, HCC1419, HCC1569 and HCC1954). The blue line indicates the threshold (established from BTRH) used to consider a cell line resistant to trastuzumab.


Analyses of HER2 , HER3 and pHER3 in the cells used in ( D ) . Numbers below the blots show the quantitation of the signal of each band , referred to SKBR3 cells ( intensity of band in SKBR3 cells = 1 ) . pHER3 levels were analyzed by a specific anti - pHER3 antibody .

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Analyses of HER2, HER3 and pHER3 in the cells used in (D). Numbers below the blots show the quantitation of the signal of each band, referred to SKBR3 cells (intensity of band in SKBR3 cells=1). pHER3 levels were analyzed by a specific anti-pHER3 antibody.


Effect of EV20 / MMAF on the cell lines used in ( D ) . Cells were counted after 5 days of treatment with the indicated doses of EV20 / MMAF .

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Effect of EV20/MMAF on the cell lines used in (D). Cells were counted after 5 days of treatment with the indicated doses of EV20/MMAF.


Knockdown of HER3 in BT474 cells . Control and HER3 - targeted short hairpin sequences were transduced in BT474 cells and HER3 , pHER3 and pAKT levels analyzed by Western . Levels of pHER3 were analyzed by a specific anti - pHER3 antibody .

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Knockdown of HER3 in BT474 cells. Control and HER3-targeted short hairpin sequences were transduced in BT474 cells and HER3, pHER3 and pAKT levels analyzed by Western. Levels of pHER3 were analyzed by a specific anti-pHER3 antibody.


Effect of HER3 knockdown on the antiproliferative action of EV20 / MMAF analyzed by cell counting after 5 days of treatment with the ADC .

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Effect of HER3 knockdown on the antiproliferative action of EV20/MMAF analyzed by cell counting after 5 days of treatment with the ADC.


Internalization of EV20 / MMAF ( 10 nM ) at indicated times in BT474 and BTRH cells . Scale bar : 20 µm . HER3 staining : red ; DAPI : blue .

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Internalization of EV20/MMAF (10 nM) at indicated times in BT474 and BTRH cells. Scale bar: 20 µm. HER3 staining: red; DAPI: blue.


Internalization of biotin - S - S - EV20 / MMAF in BT474 and BTRH cells . These cell lines were pulsed with 10 nM of biotin - S - S - EV20 / MMAF for 1 hour at 4 ° C and chased at 37 ° C at the indicated times . Subsequently , surface accessible biotin was cleaved , cells were lysed , and extracts precipitated with streptavidin - sepharose . EV20 / MMAF was analyzed by Western blotting using anti - human - HRP . Quantitative plot of the Western studies shown in ( B ) . Data are presented as the mean + SD of two independent experiments .

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Internalization of biotin-S-S-EV20/MMAF in BT474 and BTRH cells. These cell lines were pulsed with 10 nM of biotin-S-S-EV20/MMAF for 1 hour at 4°C and chased at 37°C at the indicated times. Subsequently, surface accessible biotin was cleaved, cells were lysed, and extracts precipitated with streptavidin-sepharose. EV20/MMAF was analyzed by Western blotting using anti-human-HRP. Quantitative plot of the Western studies shown in (B). Data are presented as the mean + SD of two independent experiments.


Co - localization ( yellow ) between EV20 / MMAF and LAMP1 at the indicated incubation times with EV20 / MMAF in BT474 and BTRH cells . Scale bar : 5 µm . HER3 staining : red ; LAMP1 : green , DAPI : blue .

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Co-localization (yellow) between EV20/MMAF and LAMP1 at the indicated incubation times with EV20/MMAF in BT474 and BTRH cells. Scale bar: 5 µm. HER3 staining: red; LAMP1: green, DAPI: blue.


Western blot analyses of the effect of EV20 / MMAF ( 10 nM ) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC . The same Western was probed for HER2 or HER3 expression using two differently labelled species - specific secondary fluorescent antibodies . Quantitative representation of the Western studies shown in ( E ) .

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Western blot analyses of the effect of EV20/MMAF (10 nM) on the levels of HER2 and HER3 in BT474 and BTRH cells at the indicated incubation times with the ADC. The same Western was probed for HER2 or HER3 expression using two differently labelled species-specific secondary fluorescent antibodies. Quantitative representation of the Western studies shown in (E).


Cell cycle analysis by flow cytometry of BT474 and BTRH cells ( 50 , 000 events ) treated with EV20 / MMAF ( 10 nM ) for the indicated times .

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Cell cycle analysis by flow cytometry of BT474 and BTRH cells (50,000 events) treated with EV20/MMAF (10 nM) for the indicated times.


BT474 and BTRH cells were treated for the indicated days with EV20 / MMAF ( 10 nM ) , lysed and the amount of different proteins analyzed by Western . Numbers below the blots show the quantitation of the signal of each band , referred to time 0 .

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BT474 and BTRH cells were treated for the indicated days with EV20/MMAF (10 nM), lysed and the amount of different proteins analyzed by Western. Numbers below the blots show the quantitation of the signal of each band, referred to time 0.


Action of EV20 / MMAF on mitotic spindle formation . BT474 and BTRH cells were treated ( 10 nM , 48 hours ) , fixed , stained for β - tubulin ( green ) and DAPI ( blue ) . Scale bar : 7 . 5 μm .

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Action of EV20/MMAF on mitotic spindle formation. BT474 and BTRH cells were treated (10 nM, 48 hours), fixed, stained for β-tubulin (green) and DAPI (blue). Scale bar: 7.5 μm.


Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20 / MMAF ( 10 nM , 48 hours ) in BT474 and BTRH cells . Bars represent the mean + SD of two independent experiments , calculated as follows : ( number of mitotic cells with abnormal spindles / total number of mitotic cells ) × 100 ( % ) .

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Quantitative analysis of mitotic cells with abnormal spindles treated or not with EV20/MMAF (10 nM, 48 hours) in BT474 and BTRH cells. Bars represent the mean + SD of two independent experiments, calculated as follows: (number of mitotic cells with abnormal spindles/total number of mitotic cells) ×100 (%).


Cell death induction by EV20 / MMAF ( 10 nM ) analyzed by double annexin V and PI staining in BT474 and BTRH cells ( 50 , 000 events ) .

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Cell death induction by EV20/MMAF (10 nM) analyzed by double annexin V and PI staining in BT474 and BTRH cells (50,000 events).


Time - course analysis of the effect of EV20 / MMAF ( 10 nM ) on cleaved - caspase - 3 , analyzed by Western blotting in BT474 and BTRH cells . GAPDH was used as a loading control . Samples with intensity values of 1 were used as reference .

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Time-course analysis of the effect of EV20/MMAF (10 nM) on cleaved-caspase-3, analyzed by Western blotting in BT474 and BTRH cells. GAPDH was used as a loading control. Samples with intensity values of 1 were used as reference.


Fluorimetric analyses of caspase - 3 activity in BT474 and BTRH cells treated with 10 nM EV20 / MMAF . Data show the mean + SD of triplicates of an experiment that was repeated twice .

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Fluorimetric analyses of caspase-3 activity in BT474 and BTRH cells treated with 10 nM EV20/MMAF. Data show the mean + SD of triplicates of an experiment that was repeated twice.


In vivo effect of EV20 ( 10 mg / kg , n = 4 ) , untreated control ( PBS , n = 4 ) and EV20 / MMAF ( 3 . 3 mg / kg , n = 5 ; 10 mg / kg , n = 4 ) on tumor growth in mice injected with BTRH cells . Mice were treated when tumors reached a volume of 500 mm3 . Data are represented as mean + SEM .

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In vivo effect of EV20 (10 mg/kg, n=4), untreated control (PBS, n=4) and EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4) on tumor growth in mice injected with BTRH cells. Mice were treated when tumors reached a volume of 500 mm3. Data are represented as mean + SEM.


In vivo effect of single - dose EV20 ( 10 mg / kg , n = 3 ) untreated control ( PBS , n = 4 ) , single - dose EV20 / MMAF ( 10 mg / kg , n = 4 ) or weekly trastuzumab ( 30 mg / kg , n = 4 ) on tumor growth in mice injected with BTRH # 10 cells . Data are represented as mean + SEM . In vivo effect of single - dose EV20 ( 10 mg / kg , n = 4 ) , untreated control ( PBS , n = 4 ) , single - dose EV20 / MMAF ( 10 mg / kg , n = 4 ) or weekly trastuzumab ( 30 mg / kg , n = 4 ) on tumor growth in mice injected with HCC1954 cells . Data are represented as mean + SEM .

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In vivo effect of single-dose EV20 (10 mg/kg, n=3) untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with BTRH#10 cells. Data are represented as mean + SEM. In vivo effect of single-dose EV20 (10 mg/kg, n=4), untreated control (PBS, n=4), single-dose EV20/MMAF (10 mg/kg, n=4) or weekly trastuzumab (30 mg/kg, n=4) on tumor growth in mice injected with HCC1954 cells. Data are represented as mean + SEM.


Analysis of pHistone H3 , HER3 , pHER3 and cleaved caspase - 3 in tumors of mice injected with BTRH and treated ( T ) or not ( C ) with EV20 / MMAF ( 3 . 3 mg / kg for 24 hours or two weeks ) . Numbers below the blots show the quantitation of the signal of each band , referred to that of a control untreated tumor .

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Analysis of pHistone H3, HER3, pHER3 and cleaved caspase-3 in tumors of mice injected with BTRH and treated (T) or not (C) with EV20/MMAF (3.3 mg/kg for 24 hours or two weeks). Numbers below the blots show the quantitation of the signal of each band, referred to that of a control untreated tumor.


Long - term monitoring of mice injected with BTRH cells and treated with EV20 / MMAF ( 3 . 3 mg / kg , n = 5 ; 10 mg / kg , n = 4 ) . Where indicated , mice were treated with EV20 / MMAF . Data were represented as mean + SD of the different mice groups .

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Long-term monitoring of mice injected with BTRH cells and treated with EV20/MMAF (3.3 mg/kg, n=5; 10 mg/kg, n=4). Where indicated, mice were treated with EV20/MMAF. Data were represented as mean + SD of the different mice groups.


Dose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to neratinib ( BTRN # 5 and BTRN # 24 )

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Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to neratinib (BTRN#5 and BTRN#24)


Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of ( B ) cells resistant to

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Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of (B) cells resistant to neratinib


Dose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to lapatinib ( BTRL , BTRL # 109 and BTRL # 3 )

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Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to lapatinib (BTRL, BTRL#109 and BTRL#3)


Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of cells resistant to

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Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to lapatinib


Dose - response to EV20 / MMAF after 5 days of treatment in BT474 cells resistant to T - DM1 ( BT - TDM1R # 1 and BT - TDM1R # 6 ) .

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Dose-response to EV20/MMAF after 5 days of treatment in BT474 cells resistant to T-DM1 (BT-TDM1R#1 and BT-TDM1R#6).


Levels of HER2 , pHER2 , HER3 , pHER3 , pAKT and calnexin of cells resistant to TDM1 .

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Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of cells resistant to TDM1.


Expression of indicated miRNAs in sorted naïve ( CD62L + CD44 - ) CD4 + T cells and following in vitro stimulation with anti - CD3 / anti - CD28 ( 1 - 3 days ) , relative to levels in cells prior to stimulation . Data from 3 independent experiments each using T cells pooled from 4 WT mice . Significance determined by one - way ANOVA . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Expression of indicated miRNAs in sorted naïve (CD62L+ CD44-) CD4+ T cells and following in vitro stimulation with anti-CD3 / anti-CD28 (1-3 days), relative to levels in cells prior to stimulation. Data from 3 independent experiments each using T cells pooled from 4 WT mice. Significance determined by one-way ANOVA. Data information: * p<0.05, ** p<0.01, *** p<0.001.