( 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 .

O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

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 .

O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_TYPE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O

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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

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 ) .

O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O

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 .

O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O

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 .

O O O O O B-CELL_TYPE I-CELL_TYPE O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

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 ) .

O O O O O O O O O O O B-ORGANISM B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O

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 .

O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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 .

B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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 .

O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-CELL_LINE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-CELL_LINE O O O O O O O O O O

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 .

O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

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 .

O O O O O B-ORGANISM O O B-CELL_LINE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O

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 )

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-CELL_LINE O O O B-CELL_LINE O O O

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

O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O

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 )

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-CELL_LINE O B-CELL_LINE O O O B-CELL_LINE O O O

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

O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O

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 ) .

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 .

O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O

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 .

B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Expression of indicated miRNAs in purified spleen lymphocytes ( B cells , CD4 + T cells and CD8 + T cells ) from d0 naive ( white ) and day 28 L . donovani - infected ( grey ) mice . Expression of each miRNA normalized to levels in whole naïve spleen ( dotted line ) . Data is mean + SEM of 2 experiments with cells purified from 3 - 5 pooled spleens .

B-EXP_ASSAY O O O O O B-TISSUE B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-DISEASE O O O B-ORGANISM O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O

Expression of indicated miRNAs in purified spleen lymphocytes (B cells, CD4+ T cells and CD8+ T cells) from d0 naive (white) and day 28 L. donovani-infected (grey) mice. Expression of each miRNA normalized to levels in whole naïve spleen (dotted line). Data is mean + SEM of 2 experiments with cells purified from 3-5 pooled spleens.


Volcano plot of RNAseq gene expression in splenic WT and miR - 132 - / - CD4 + T cells from d28 L . donovani infected mice . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 5 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - ( p < 0 . 05 ) are shown in red . Dotted box indicates transcripts significantly up - regulated in miR - 132 - / - CD4 + T cells by more than 50 % .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O

Volcano plot of RNAseq gene expression in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 5 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- (p<0.05) are shown in red. Dotted box indicates transcripts significantly up-regulated in miR-132-/- CD4+ T cells by more than 50%.


STRING network analysis of significantly up - regulated transcripts in CD4 + T cells from spleen of d28 L . donovani infected miR - 132 - / - mice compared to WT cells . Cluster of ribosomal proteins shown in green circle , with coding RP transcripts ( black ) and pseudogenes ( red ) indicated . Secondary clusters are shown in grey .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

STRING network analysis of significantly up-regulated transcripts in CD4+ T cells from spleen of d28 L. donovani infected miR-132-/- mice compared to WT cells. Cluster of ribosomal proteins shown in green circle, with coding RP transcripts (black) and pseudogenes (red) indicated. Secondary clusters are shown in grey.


Volcano plot of all RP genes in splenic WT and miR - 132 - / - CD4 + T cells from d28 L . donovani infected mice . RPL genes are shown as circles , RPS genes as triangles , and pseudogenes as squares . Red symbols indicate significant difference between WT and miR - 132 - / - cells ( p < 0 . 05 ) whereas black non - significant .

O O O O O O O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

Volcano plot of all RP genes in splenic WT and miR-132-/- CD4+ T cells from d28 L. donovani infected mice. RPL genes are shown as circles, RPS genes as triangles, and pseudogenes as squares. Red symbols indicate significant difference between WT and miR-132-/- cells (p<0.05) whereas black non-significant.


Expression of RP transcripts determined by qPCR from L . donovani infected d28 WT ( blue ) and miR - 132 - / - mice ( red ) . N = 9 for each WT and miR - 132 - / - from 2 independent infection experiments . Box extends from 25 - 75th percentile , whiskers are minimum and maximum values , and horizontal lines indicate median . Significance determined by unpaired t - test . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of RP transcripts determined by qPCR from L. donovani infected d28 WT (blue) and miR-132-/- mice (red). N=9 for each WT and miR-132-/- from 2 independent infection experiments. Box extends from 25-75th percentile, whiskers are minimum and maximum values, and horizontal lines indicate median. Significance determined by unpaired t-test. Data information: * p<0.05, ** p<0.01, *** p<0.001.


Fold change of all RP transcripts ( grey ) in Th1 cells compared to naïve CD4 + T cells . Data taken from RNA sequencing experiments described in reference 24 . Fold changes in IL - 10 ( red ) and IFNγ ( blue ) indicated for comparison . The statistical significance of the observed up - regulation of RP transcripts in Th1 cells is determined by Chi - squared test .

O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O

Fold change of all RP transcripts (grey) in Th1 cells compared to naïve CD4+ T cells. Data taken from RNA sequencing experiments described in reference 24. Fold changes in IL-10 (red) and IFNγ (blue) indicated for comparison. The statistical significance of the observed up-regulation of RP transcripts in Th1 cells is determined by Chi-squared test.


A . Volcano plot ( Log2 ( Fold Change ) vs - Log ( P value ) ) of RNA gene expression in purified naïve CD62L + CD44 - WT and miR - 132 - / - CD4 + T cells . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 4 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - cells ( p < 0 . 05 ) shown in red .

O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

A. Volcano plot (Log2(Fold Change) vs -Log(P value)) of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.


B . Volcano plot of RNA gene expression in purified naïve CD62L + CD44 - WT and miR - 132 - / - CD4 + T cells following 18hr in vitro stimulation with anti - CD3 / anti - CD28 under Th1 conditions . Fold change determined as log2 mean FPKM ( miR - 132 - / - / WT ) from 4 WT and 4 miR - 132 - / - mice . Transcripts significantly different between WT and miR - 132 - / - cells ( p < 0 . 05 ) shown in red .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

B. Volcano plot of RNA gene expression in purified naïve CD62L+ CD44- WT and miR-132-/- CD4+ T cells following 18hr in vitro stimulation with anti-CD3/anti-CD28 under Th1 conditions. Fold change determined as log2 mean FPKM (miR-132-/-/WT) from 4 WT and 4 miR-132-/- mice. Transcripts significantly different between WT and miR-132-/- cells (p<0.05) shown in red.


C . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in naïve CD4 + T cells from WT or miR - 132 - / - mice .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O

C. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in naïve CD4+ T cells from WT or miR-132-/- mice.


D . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in in vitro polarised ( Th1 condtions , 18h post stimulation ) CD4 + T cells from WT or miR - 132 - / - mice .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O

D. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in in vitro polarised (Th1 condtions, 18h post stimulation) CD4+ T cells from WT or miR-132-/- mice.


E . Volcano plot of transcripts containing a conserved miR - 212 / 132 - 3p target site in spleen CD4 + T cells from d28 L . donovani infected WT or miR - 132 - / - mice .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O

E. Volcano plot of transcripts containing a conserved miR-212/132-3p target site in spleen CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice.


F . BTAF1 transcript levels determined by qRTPCR in WT ( blue ) or miR - 132 - / - ( red ) in naïve ( d0 ) and Th1 polarised for 18h ( d1 ) CD4 + T cells , and CD4 + T cells from d28 L . donovani infected WT or miR - 132 - / - mice . N = 8 - 9 for each WT and miR - 132 - / Data information : Significance in ( F ) determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 .

O O B-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. BTAF1 transcript levels determined by qRTPCR in WT (blue) or miR-132-/- (red) in naïve (d0) and Th1 polarised for 18h (d1) CD4+ T cells, and CD4+ T cells from d28 L. donovani infected WT or miR-132-/- mice. N=8-9 for each WT and miR-132-/ Data information: Significance in (F) determined by unpaired t-test. * p<0.05, ** p<0.01.


G . Expression of BTAF1 protein in d0 naïve and d1 ( 18hr ) Th1 - polarised WT and miR - 132 - / - CD4 + T cells , as determined by Western blot . Each lane from individual mouse , and representative of two independent experiments .

O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O

G. Expression of BTAF1 protein in d0 naïve and d1 (18hr) Th1-polarised WT and miR-132-/- CD4+ T cells, as determined by Western blot. Each lane from individual mouse, and representative of two independent experiments.


H . Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3 ' UTR ( white ) or BTAF1 3 ' UTR on which the miR - 132 binding site is mutated ( grey ) downstream of renilla luciferase , in the presence of miR - 132 - 3p or miR - 212 - 3p mimics . Error bars indicate SEM from eight replicate treatments . Data information : Significance in ( H ) determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Relative luciferase activity in HeLa transfected with plasmid containing WT BTAF1 3'UTR (white) or BTAF1 3'UTR on which the miR-132 binding site is mutated (grey) downstream of renilla luciferase, in the presence of miR-132-3p or miR-212-3p mimics. Error bars indicate SEM from eight replicate treatments. Data information: Significance in (H) determined by unpaired t-test. * p<0.05, ** p<0.01.


A . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non - targeting control ( NTC ) mimics ( white ) or miR - 132 - 3p mimics ( grey ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with Non-targeting control (NTC) mimics (white) or miR-132-3p mimics (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


B . p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR - 132 - 3p mimics determined by Western blot . GAPDH was used as a loading control . Right panel indicates mean + SEM of 4 experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. p300 and BTAF1 protein levels in MEF transfected with NTC mimics or miR-132-3p mimics determined by Western blot. GAPDH was used as a loading control. Right panel indicates mean + SEM of 4 experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


C . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA ( white ) or p300 siRNA ( grey ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or p300 siRNA (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


D . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA ( white ) or BTAF1 siRNA ( grey ) . Data information : Statistical significance is determined by unpaired t -

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

D. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or BTAF1 siRNA (grey). Data information: Statistical significance is determined by unpaired t-test


E . mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR - 132 - 3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h . Levels are normalised to cells transfected with NTC siRNA and NTC mimic . Data information : Statistical significance is determined by unpaired t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC or miR-132-3p mimics and NTC siRNA or p300 or BTAF1 siRNAs for 48h. Levels are normalised to cells transfected with NTC siRNA and NTC mimic. Data information: Statistical significance is determined by unpaired t-test * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.


F . Puromycin incorporation ( following 10 - minute pulse and 50 - minute chase ) determined by western blot in MEFs transfected with NTC or miR - 132 - 3p or miR - 212 - 3p mimics . Data information : Statistical significance is determined by unpaired t -

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

F. Puromycin incorporation (following 10-minute pulse and 50-minute chase) determined by western blot in MEFs transfected with NTC or miR-132-3p or miR-212-3p mimics. Data information: Statistical significance is determined by unpaired t-test


A . Percentage of IFNγ + live TCRβ + CD4 + cells from L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice , determined by intracellular cytokine staining . Data representative of 3 independent experiments with 3 - 5 mice per group . Data information : statistical significance was determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant .

O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Percentage of IFNγ+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.


B . Percentage of IFNγ + / IL - 10 + live TCRβ + CD4 + cells from L . donovani infected WT ( blue ) or miR - 132 - / - ( red ) mice , determined by intracellular cytokine staining . Data representative of 3 independent experiments with 3 - 5 mice per group . Data information : statistical significance was determined by unpaired t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . NS : not significant .

O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM B-DISEASE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Percentage of IFNγ+/IL-10+ live TCRβ+ CD4+ cells from L. donovani infected WT (blue) or miR-132-/- (red) mice, determined by intracellular cytokine staining. Data representative of 3 independent experiments with 3-5 mice per group. Data information: statistical significance was determined by unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. NS: not significant.