( C ) lysate from BMDMs that were treated with IFN - γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 UBQLN2 .

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(C) Cellular lysate from BMDMs that were treated with IFN-γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2.


( D ) BMDMs were transfected with siRNAs targeting UBQLN1 or a non - targeting control ( siCON ) lysates were analyzed by WB using an antibody that recognizes UBQLN1 UBQLN2 . ( C - D ) Actin served as a loading control .

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(D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control.


( E ) BMDMs were infected with Mtb live / dead treated with AnTc 24h prior to fixation .

(E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation.


( F ) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON infected with live / dead Mtb . IFN - γ was added 1d prior to infection if indicated . % Viable were quantified 24 48 hpi . Data are from 2 independent experiments . * P < 0 . 05 ; * * * P < 0 . 001 , Fisher ' s exact , comparing proportion of viable non - viable in indicated samples .

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(F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-γ was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *P<0.05; ***P<0.001, Fisher's exact test, comparing the proportion of viable and non-viable bacteria in indicated samples.


( G ) BMDMs were treated with indicated siRNAs three days prior to infection then activated with IFN - γ one day prior to infection with H37Rv or ΔesxA . CFU were enumerated 4 96 hpi . * P < 0 . 05 , unpaired Student ' s t - . Data are representative of three independent experiments .

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(G) BMDMs were treated with the indicated siRNAs three days prior to infection and then activated with IFN-γ one day prior to infection with H37Rv or ΔesxA. CFU were enumerated 4 and 96 hpi. *P< 0.05, unpaired Student's t-test. Data are representative of three independent experiments.


( H ) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb ( H37Rv ) IFN - γ was measured by

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(H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) and IFN-γ was measured by ELISA


( I ) UBQLN1 - silenced BMDMs controls were infected with H37Rv or ΔesxA . ( H - I ) 24 hpi infected BMDMs were co - cultured with P25TCR - Tg CD4 + T , IFN - γ was measured by ELISA . Data is representative of 3 independent experiments . * P < 0 . 05 , * * P < 0 . 005 , unpaired Student ' s t - . ( B , F , Γ , H ) Results are mean + / - ; ns , not significant .

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(I) UBQLN1-silenced BMDMs and controls were infected with H37Rv or ΔesxA. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-γ was measured by ELISA. Data is representative of 3 independent experiments. *P<0.05, **P<0.005, unpaired Student's t-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.


( A ) BMDMs treated with IFN - γ infected for 24h with GFP - Mtb were immunostained for UBQLN1 either ubiquitin ( using FK2 antibody ) or p62 . GFP - LC3 BMDMs infected with DsRed - Mtb were immunostained for UBQLN1 . ( B ) of those Mtb that co - localize with at least one of markers shown in A . More than 375 total were counted in two independent experiments , number co - localizing with at least one marker is indicated as " total " under each bar .

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(A) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as "total" under each bar.


( C ) Wt macrophages ( Atg16L1flox / flox ; Cre - ) autophagy - deficient macrophages ( Atg16L1flox / flox Lyz - cre ; Cre + ) were activated with IFN - γ or not , followed by infection with live / dead Mtb reporter . % viable was quantified after addition of AnTc . * * P < 0 . 005 , * * * P < 0 . 0001 , Fisher ' s exact ( in which proportions of viable to non - viable Mtb was compared ) .

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(C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared).


( D ) Atg16L1flox / flox Cre + Cre - BMDMs treated with IFN - γ infected for 24h with GFP - Mtb were immunostained for UBQLN1 p62 . number of Mtb co - localizating with markers is shown . ( C - D ) At least 100 were analyzed in three independent experiments .

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(D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments.


( E ) from IFN - γ treated BMDMs of indicated genotype infected with GFP - Mtb immunostained with UBQLN1 p62 . Arrows indicates p62 + UBQLN1 + Mtb .

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(E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.


( A - C ) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA . show IFN - γ treated macrophages infected with GFP - Mtb ( A - B ) or DsRed - Mtb ( C ) for 24h . were immunostained for ubiquitin ( Ub ) with FK2 antibody ( A ) or p62 ( B ) , which are shown in red . ( C ) GFP - LC3 BMDMs were infected with DsRed - Mtb . LC3 is shown in green , whereas Mtb are red . ( A - C ) shows of Mtb that colocalized with indicated marker in macrophages that were treated with IFN - γ as indicated . Results are mean + / - from two independent experiments , with at least 100 analyzed per experiment . * * * P < 0 . 001 , Fisher ' s exact ( in which proportion of Mtb associated with marker in a given condition was compared to proportion found in sample treated with siCON IFN - γ ) .

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(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-γ treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-γ as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***P<0.001, Fisher's exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-γ).


( A ) WT Park2 - / - BMDMs were pretreated with IFN - γ , infected with GFP - Mtb for 24h , immunostained with FK2 antibody . colocalization with FK2 immunoreactivity was quantified from 2 independent experiments .

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(A) WT and Park2-/- BMDMs were pretreated with IFN-γ, infected with GFP-Mtb for 24h, and immunostained with the FK2 antibody. Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments.


( B ) Park2 - / - BMDMs pretreated or not treated with IFN - γ were infected with GFP - Mtb for 24h immunostained with UBQLN1 specific antibody ; Arrows indicate Mtb co - localized with UBQLN1 . ns - not significant .

(B) Park2-/- BMDMs pretreated or not treated with IFN-γ were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant.


( C ) Park2 - / - BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection , treated with IFN - γ , infected with GFP - Mtb for 24h prior to immunostaining for ubiquitin ( FK2 antibody ) . Ubiquitin ( FK2 + ) were quantified from two independent experiments . ( A , C ) * * P < 0 . 01 , Fisher ' s exact ( comparing proportion of FK2 positive in indicated samples ) . Results are mean + / - . A minimum of 100 Mtb were counted in each experiment .

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(C) Park2-/- BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection, treated with IFN-γ, and infected with GFP-Mtb for 24h prior to immunostaining for ubiquitin (FK2 antibody). Ubiquitin (FK2+) bacteria were quantified from two independent experiments. (A, C) **P<0.01, Fisher's exact test (comparing the proportion of FK2 positive bacteria in indicated samples). Results are mean +/- SEM. A minimum of 100 Mtb were counted in each experiment.


B . Vero E6 were transfected with 10 nM of siRNA before infection by SARS - CoV - 2 at a multiplicity of infection ( MOI ) of 0 . 1 after 24 h . numbers of viral copies were quantified with RT - qPCR . negative control siRNA served as control is abbreviated as " Ctrl . " C1 - C11 represent final candidate sequences after selection . siRNAs capable of inhibiting up to 99 % of viral envelope gene expression with P values < 0 . 005 compared with Ctrl siRNA are marked with * . P value by Student t .

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B. Vero E6 cells were transfected with 10 nM of siRNA before infection by SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 after 24 h. The numbers of viral RNA copies were quantified with RT-qPCR. The negative control siRNA served as the control and is abbreviated as "Ctrl." C1-C11 represent the final candidate sequences after selection. The siRNAs capable of inhibiting up to 99% of viral envelope gene expression with P values < 0.005 compared with Ctrl siRNA are marked with *. P value by Student t test.


E . IC50 of C6 fully modified C6G25S . Vero E6 were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6 or C6G25S before infection at an MOI of 0 . 1 . viral was quantified by RT - qPCR at 24 h after infection . F . IC50 data for viral RdRp inhibition by C6G25S . Vero E6 were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before infection at an MOI of 0 . 1 . viral was quantified by RT - qPCR at 24 h after infection .

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E. IC50 of C6 and the fully modified C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6 or C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. F. IC50 data for viral RdRp inhibition by C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. D


B . IC50 for C6G25S against different variants . Vero E6 were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before infection with different strains of SARS - CoV - 2 . viral was quantified by RT - qPCR at 24 h after infection . Data are presented as mean ± SD of three biological replicates .

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B. IC50 for C6G25S against different variants. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before infection with different strains of SARS-CoV-2. The viral RNA was quantified by RT-qPCR at 24 h after infection. Data are presented as mean ± SD of three biological replicates.


A , B , C . K18 - hACE2 - transgenic mice treated with C6G25S by 1 . 48 mg / L of AI for 30 min ( A ) , 50 μL of saline containing 50 μg of C6G25S by IN ( B ) , or PBS ( C ) ( n = 5 per group ) . C6G25S distribution in lungs was visualized by in situ hybridization ( ISH ) with C6G25S - specific probe ( red color ) . Bronchi ( i ) bronchioles ( ii ) marked with boxes are enlarged on right .

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A, B, C. K18-hACE2-transgenic mice treated with C6G25S by 1.48 mg/L of AI for 30 min (A), 50 μL of saline containing 50 μg of C6G25S by IN (B), or PBS (C) (n = 5 per group). C6G25S distribution in lungs was visualized by in situ hybridization (ISH) staining with C6G25S-specific probe (red color). Bronchi (i) and bronchioles (ii) marked with the boxes are enlarged on the right.


E , F . C57 / B6 mice ( n = 3 per group ) were treated by AI with C6G25S 1 . 48 mg / L for 30 min ( E ) or 50 μL of saline containing 50 μg of C6G25S by IN administration ( F ) . C6G25S deposited in whole lungs nasal cavities was quantified after whole tissue homogenization followed by - loop RT - qPCR . data represent mean ± SD . P value by Student t .

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E, F. C57/B6 mice (n = 3 per group) were treated by AI with C6G25S 1.48 mg/L for 30 min (E) or 50 μL of saline containing 50 μg of C6G25S by IN administration (F). The C6G25S deposited in whole lungs and nasal cavities was quantified after whole tissue homogenization followed by stem-loop RT-qPCR. Quantification data represent mean ± SD. P value by Student t test.


A . K18 - hACE2 - transgenic mice ( Winkler et al . , 2020 ) were treated once daily for 3 days before intranasal challenge with 104 plaque - forming units ( PFU ) of original . Prophylactic treatment consists of 30 min of AI ( 1 . 48 mg / l of C6G25S ) , followed by IN of 50 μg C6G25S . Mice receiving vehicle control ( saline ) for both AI IN are annotated as control . Viral ( left ) infectious virions ( right ) in lungs were quantified with RT - qPCR plaque forming , respectively , at 2 days post - infection ( dpi ) . B . Mice were challenged intranasally with 104 PFU of co - treatment with 1 . 48 mg / L of C6G25S or vehicle control ( saline ) by AI for 30 min on day 0 ( right after infection ) day 1 . Viral infectious virions were quantified at 2 dpi .

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A. K18-hACE2-transgenic mice (Winkler et al., 2020) were treated once daily for 3 days before intranasal challenge with 104 plaque-forming units (PFU) of the original virus. Prophylactic treatment consists of 30 min of AI (1.48 mg/l of C6G25S), followed by IN of 50 μg C6G25S. Mice receiving vehicle control (saline) for both AI and IN are annotated as control. Viral RNA (left) and infectious virions (right) in lungs were quantified with RT-qPCR and plaque forming assay, respectively, at 2 days post-infection (dpi). B. Mice were challenged intranasally with 104 PFU of virus and co-treatment with 1.48 mg/L of C6G25S or vehicle control (saline) by AI for 30 min on day 0 (right after infection) and day 1. Viral RNA and infectious virions were quantified at 2 dpi. C


A . Immunohistochemical ( ) of viral spike in lung sections from K18 - hACE2 mice ( Winkler et al . , 2020 ) at day 5 post - infection . Spike were detected with anti - spike antibody stained brown . of bronchial epithelium of vehicle control treated ( i ii ) C6G25S treated ( iii iv ) , alveoli of vehicle control treated ( v vi ) C6G25S treated ( vii viii ) , bronchiole blood vessel of vehicle control treated ( ix x ) C6G25S treated ( xi xii ) group were shown . Syncytial was indicated by green arrow in ( xi ) thrombosis in ( x ) .

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A. Immunohistochemical (IHC) staining of viral spike proteins in lung sections from K18-hACE2 mice (Winkler et al., 2020) at day 5 post-infection. Spike proteins were detected with anti-spike antibody and stained brown. Images of bronchial epithelium of vehicle control treated (i and ii) and C6G25S treated (iii and iv), alveoli of vehicle control treated (v and vi) and C6G25S treated (vii and viii), bronchiole and blood vessel of vehicle control treated (ix and x) and C6G25S treated (xi and xii) group were shown. Syncytial cell was indicated by green arrow in (xi) and thrombosis in (x).


B . ISH of viral in lungs of vehicle control group ( left ) C6G25S treated group ( right ) at day 2 post - infection . Viral was stained brown as green arrows indicated . are representative of 5 for each group .

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B. ISH staining of viral RNA in lungs of vehicle control group (left) and C6G25S treated group (right) at day 2 post-infection. Viral RNA was stained brown as green arrows indicated. Images are representative of 5 animals for each group.


C . of of Ly6G + ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) C6G25S treated group ( right ) at day 2 post - infection .

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C. Images of IHC staining of Ly6G+ Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection.


D . of of F4 / 80 + ( Brown color ) in lungs of vehicle control ( left ) C6G25S treated group ( right ) at day 2 post - infection . E . of of CD3 + T ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) C6G25S treated group ( right ) at day 2 post - infection .

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D. Images of IHC staining of F4/80+ Cells (Brown color) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. E. Images of IHC staining of CD3 + T Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. F


Representative WBs of RPE - 1 wild - type ( WT ) two selected KO clones for AKAP450 ( akap KO ) , PCNT ( pcnt KO ) CDK5Rap2 ( c5rap2 KO ) probed with indicated antibodies . GM130 was used as a loading

Representative WBs of RPE-1 wild-type (WT) cells and the two selected KO clones for AKAP450 (akap KO), PCNT (pcnt KO) and CDK5Rap2 (c5rap2 KO) probed with the indicated antibodies. GM130 was used as a loading control


Confocal IF of RPE - 1 WT , single KO ( akap KO , pcnt KO c5rap2 KO ) double KO ( pc - ak - 2KO , left panels ; pc - c5 - 2KO , right panels ) stained with indicated antibodies . yellow dashed indicates nucleus contour ( N ) arrows point centrosome ( CTR ) . GA marks Golgi Apparatus . Scale bars : 5 μm .

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Confocal IF images of RPE-1 WT, single KO (akap KO, pcnt KO and c5rap2 KO) and double KO (pc-ak-2KO, left panels; pc-c5-2KO, right panels) cells stained with the indicated antibodies. The yellow dashed line indicates the nucleus contour (N) and the arrows point the centrosome (CTR). GA marks the Golgi Apparatus. Scale bars: 5 μm.


Representative WB of RPE - 1 WT two selected PCNT / AKAP450 ( pc - ak - 2KO ) PCNT / CDK5Rap2 ( pc - c5 - 2KO ) double knockout mutated clones probed with indicated antibodies . GM130 was used as a loading

Representative WB of RPE-1 WT cells and the two selected PCNT/AKAP450 (pc-ak-2KO) and PCNT/CDK5Rap2 (pc-c5-2KO) double knockout mutated clones probed with the indicated antibodies. GM130 was used as a loading control


Representative WB of RPE - 1 WT indicated single or double KO lines probed with anti - AKAP450 , PCNT CDK5Rap2 antibodies ( left ) . of signal from three independent WB experiments is shown at right . # indicates KO . arrow points dramatic decrease of CDK5Rap2 in pc - ak - 2KO . Hsp70 was used as a loading

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Representative WB of RPE-1 WT cells and the indicated single or double KO cell lines probed with anti-AKAP450, PCNT and CDK5Rap2 antibodies (left). Quantification of signal intensity from three independent WB experiments is shown at right. # indicates KO proteins. The arrow points the dramatic decrease of CDK5Rap2 levels in pc-ak-2KO cell line. Hsp70 was used as a loading control


Confocal of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO pc - c5 - 2KO stained for EB1 ( green ) centrosomal CAP350 ( red ) . High magnification single - channel of selected areas are shown at right . Scatter plot showing of EB1 comets from three independent experiments as that shown in A . A region of interest of 3 μm radius around CAP350 signal was used to number of comets . Horizontal black lines represent mean .

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Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) and the centrosomal protein CAP350 (red). High magnification single-channel images of selected areas are shown at right. Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A. A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets. Horizontal black lines represent the mean.


MT regrowth experiments of WT mixed with either WT ( as a control ) , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO or pc - c5 - 2KO . Samples were fixed 3 min after NZ washout stained for EB1 ( green ) indicated in red channel . of EB1 signal around

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MT regrowth experiments of WT cells mixed with either WT (as a control), akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO or pc-c5-2KO cells. Samples were fixed 3 min after NZ washout and stained for EB1 (green) and the indicated protein in the red channel. Quantification of EB1 signal intensity around the centrosome


WT pc - ak - 2KO were transfected with a combination of three siRNAs against CEP192 , fixed triple labelled for EB1 , CEP192 CAP350 , as a centrosomal marker . Scramble siRNA was used as a control .

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WT and pc-ak-2KO cells were transfected with a combination of three siRNAs against CEP192, fixed and triple labelled for EB1, CEP192 and CAP350, as a centrosomal marker. Scramble siRNA was used as a control.


Confocal of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO pc - c5 - 2KO stained for EB1 ( green ) EB1 comets around

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Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) EB1 comets around the centrosome


Three min MT regrowth experiments in WT pc - ak - 2KO transfected with either scramble or CEP192 siRNAs .

Three min MT regrowth experiments in WT and pc-ak-2KO cells transfected with either scramble or CEP192 siRNAs.


Quantification of the EB1 signal intensity around the centrosome


MT regrowth experiments in WT c5rap2 KO or pcnt KO . were stained as indicated . Arrows point centrosome .

MT regrowth experiments in WT and c5rap2 KO or pcnt KO cells. Cells were stained as indicated. Arrows point the centrosome.


Quantification of EB1 signal intensity at Golgi membranes.


WB of AKAP450 co - immunoprecipitation assays revealed with indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant .

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WB of AKAP450 co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.


WB of AKAP450 PCNT ( B ) co - immunoprecipitation assays revealed with indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant .

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WB of AKAP450 and PCNT (B) co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.


Centrosomes from WT , single double KO , as indicated , were labelled for AKAP450 PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around centrosome was determined normalized to WT mean .

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Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.


Centrosomes from WT , single double KO , as indicated , were labelled for CDK5Rap2 Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around centrosome was determined normalized to WT mean .

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Centrosomes from WT, single and double KO cells, as indicated, were labelled for CDK5Rap2 Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.


Centrosomes from WT , single double KO , as indicated , were labelled for γ - tubulin ( G ) . Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around centrosome was determined normalized to WT mean .

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Centrosomes from WT, single and double KO cells, as indicated, were labelled for γ-tubulin (G). Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.


WT pc - ak - 2KO were transfected with either scramble or CEP192 siRNAs , fixed stained for γ - tubulin . of is show at right .

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WT and pc-ak-2KO cells were transfected with either scramble or CEP192 siRNAs, fixed and stained for γ-tubulin. Quantification of the fluorescence intensity is show at right.


IF of WT pcnt KO RPE - 1 treated with NZ for 3h triple stained for AKAP450 ( green ) , CDK5Rap2 ( red ) GMAP210 as a Golgi marker ( blue ) . At right , of co - localization of either AKAP450 or CDK5Rap2 with Golgi marker GMAP210 in WT pcnt KO .

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IF images of WT and pcnt KO RPE-1 cells treated with NZ for 3h and triple stained for AKAP450 (green), CDK5Rap2 (red) and GMAP210 as a Golgi marker (blue). At right, quantification of fluorescence co-localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells.


Control centrinone - treated were subjected to a 3 - min MT regrowth , fixed labelled for EB1 ( green ) giantin ( red ) . Insets show magnified of boxed regions .

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Control and centrinone-treated cells were subjected to a 3-min MT regrowth assay, fixed and labelled for EB1 (green) and giantin (red). Insets show magnified images of boxed regions.


of total number of growing MTs per ( left ) , number of MTs nucleated per Golgi fragment ( middle ) number of MT - nucleating Golgi fragments ( right ) in both control centrosome - less RPE - 1 . Scatter plots show each data point . Horizontal black lines bars represent mean .

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Quantification of the total number of growing MTs per cell (left), the number of MTs nucleated per Golgi fragment (middle) and the number of MT-nucleating Golgi fragments (right) in both control and centrosome-less RPE-1 cells. Scatter plots show each individual data point. Horizontal black lines bars represent the mean.


RPE - 1 were treated wi th centrinone for either 7 days ( left ) or for 5 days then allowed to recover for 48h in absence of drug prior to fixation ( right ) . Representative of a triple IF with EB1 ( green ) , γ - tubulin ( red ) giantin ( blue ) as a Golgi marker are shown . Insets show MT nucleation from Golgi elements ( left ) or from multiple centrioles ( right ) are enlarged at right . Arrowheads indicate position of Golgi elements .

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RPE-1 cells were treated with centrinone for either 7 days (left) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation (right). Representative images of a triple IF staining with EB1 (green), γ-tubulin (red) and giantin (blue) as a Golgi marker are shown. Insets show MT nucleation from Golgi elements (left) or from multiple centrioles (right) and are enlarged at right. Arrowheads indicate the position of Golgi elements.


of of exhibiting Golgi - nucleating activity in centrinone - treated 48h after washout of drug . were divided into two categories depending on number of centrioles that were visualized by labelling for centriole Cep63 . At least 100 from two independent experiments were analyzed in each condition .

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Quantification of the percentage of cells exhibiting Golgi-nucleating activity in centrinone-treated cells 48h after the washout of the drug. Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63. At least 100 cells from two independent experiments were analyzed in each condition.


Control ( top ) centrinone - treated ( bottom ) RPE - 1 double stained for either AKAP450 , CDK5Rap2 , PCNT or CEP192 ( shown in green ) giantin ( red ) as a Golgi marker . was counterstained with DAPI is shown in blue . Single channel ( left , green ) merged ( right ) are shown in each case . arrows indicate centrosome ( CTR ) .

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Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR).


IF of control centrinone - treated RPE - 1 treated with NZ to induce GA fragmentation double stained with either anti - AKAP450 , anti - CDK5Rap2 or anti - PCNT ( shown in green ) anti - giantin antibodies ( ) . Enlarged views of boxed areas are presented at bottom with or without signal of Golgi marker . yellow dashed indicates contour of nucleus . Arrowheads point to accumulation of respective in Golgi surfaces .

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IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces.


Box - - whisker plots showing of association between indicated Golgi elements Top bottom ends of boxes represent 75th 25th percentiles , whiskers represent 90th 10th percentiles . median is depicted with a solid . Golgi elements from at least 14 were delineated ( > 500 elements / experiment ) of each spot associated with each Golgi element was measured . Data were collected from two independent experiments normalized to WT mean .

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Box-and-whisker plots showing quantification of association between the indicated protein and Golgi elements Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Individual Golgi elements from at least 14 cells were delineated (>500 elements/experiment) and the intensity of each protein spot associated with each Golgi element was measured. Data were collected from two independent experiments and normalized to WT mean.


MT - regrowth experiment after NZ treatment in centrinone - treated . High magnification at bottom are representative of a triple with α - tubulin ( green ) , GM130 ( blue ) indicated ( red ) show MTs growing from surface of GA . Double labelling without α - tubulin are also shown ( top ) to allow better visualization of PCM - spots on Golgi elements acting as MT nucleation sites ( arrowheads ) .

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MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads).


Centrinone - treated pcnt KO , c5rap2 KO akap KO , were incubated with NZ for 3h , fixed double labelled for GM130 either CDK5Rap2 or PCNT as indicated ( n = 3 ) .

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Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3).


MT regrowth in centrinone - treated WT , pcnt KO akap KO RPE - 1 stained with antibodies to EB1 giantin . At right , of EB1 at Golgi membranes in WT pcnt KO treated with centrinone , as a measure of MT - nucleation from GA . Data were collected from two independent experiments normalized to WT mean .

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MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.


Representative MT network of a centrosome - less akap KO .

Representative MT network of a centrosome-less akap KO cell.


MT - regrowth experiment after NZ treatment in centrinone - treated akap KO . A representative confocal image of a double labelling for EB1 ( green ) giantin ( blue ) is shown . High magnification at right shows that growing MT asters are not associated to Golgi fragments .

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MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments.


Centrinone - treated akap KO were subjected to a MT regrowth double stained with indicated antibodies . High magnification of boxed areas are shown as merge ( top ) or green or red labelling ( middle bottom panels ) .

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Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels).


lacking AKAP450 were treated with centrinone in order to induce formation of then transfected with siRNAs specific for CDK5Rap2 ( top panels ) or PCNT ( bottom panels ) . After 3 - min MT regrowth , were fixed stained with indicated antibodies .

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Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies.


MT regrowth experiments in centrinone - treated akap KO transfected with either scramble or PCNT siRNAs . After a 3h NZ treatment , MTs were allowed to polymerize , at indicated time points after washout , were fixed labelled for α - tubulin ( green ) , PCNT ( red ) CAP350 ( blue ) .

MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue).


MT repolymerization experiments in centrinone - treated akap KO transfected with either scramble or PCNT siRNAs . After NZ treatment removal , were incubated for 10 min on ice then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin . were finally fixed triple stained with EB1 ( green ) , PCNT ( red ) CAP350 ( blue ) antibodies . Enlarged views of boxed areas are presented at right . Scale bars , 5 μm .

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MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.


Confocal showing MT network in WT , akap KO , c5rap2 KO , pcnt KO pc - ak - 2KO RPE - 1 fixed stained with antibodies against α - tubulin ( left ) or EB1 ( right ) . red yellow lines indicate contour of .

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Confocal images showing MT network in WT, akap KO, c5rap2 KO, pcnt KO and pc-ak-2KO RPE-1 cells fixed and stained with antibodies against α-tubulin (left) or EB1 (right). The red and yellow lines indicate the contour of cells.


Confocal showing MT network in centrinone - treated WT , akap KO , c5rap2 KO pcnt KO backgrounds . containing extra centrosomes induced by centrinone washout were also quantified ( bottom panels ) .

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Confocal images showing MT network in centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO backgrounds. Cells containing extra centrosomes induced by centrinone washout were also quantified (bottom panels).


of α - tubulin ( as a measure of MT mass polymer , C ) , in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of α-tubulin fluorescence intensity (as a measure of MT mass polymer, C), in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


of number of EB1 comets per ( D ) in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of number of EB1 comets per cell (D) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


of α , ( E ) in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of α , cell area (E) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


of MT density ( MT mass polymer / ) ( F ) in control centrinone - treated WT , akap KO , c5rap2 KO pcnt KO . Non - centrinone treated double KO centrinone - treated WT after washout were also included . Scatter plots show each data point horizontal black lines bars represent mean .

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Quantification of MT density (MT mass polymer/cell area) (F) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.


A Bisulfite methylation sequencing of Athila6ATE transcriptional start site ( TSS ) in genotypes with silenced TEs ( left of dashed ) or transcriptionally active TEs ( right of dashed ) . Methylation in CG ( red ) , CHG ( blue ) CHH ( green ) contexts are shown ( H = C , T , or A ) . Error bars represent Wilson score interval 95 % confidence limits . For multiple statistical , one ‐ way ANOVA was performed using Tukey ' s multiple comparison . ns , not significant .

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A Bisulfite DNA methylation sequencing of the Athila6ATE transcriptional start site (TSS) in genotypes with silenced TEs (left of the dashed line) or transcriptionally active TEs (right of the dashed line). Methylation in the CG (red), CHG (blue) and CHH (green) sequence contexts are shown (H = C, T, or A). Error bars represent Wilson score interval 95% confidence limits. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test. ns, not significant.


B reads ‐ per ‐ million ‐ ( RPM ) ‐ normalized accumulation of Athila6ATE siRNAs from TE ‐ silenced epigenomes ( left ) ddm1TE ‐ active epigenomes ( right ) . Inset graphs reflecting same values are shown for select genotypes . graphs have same y ‐ axis scale are shown for increased resolution in genotypes with low siRNA .

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B The reads‐per‐million‐(RPM)‐normalized accumulation of Athila6ATE siRNAs from TE‐silenced epigenomes (left) and ddm1TE‐active epigenomes (right). Inset line graphs reflecting the same values are shown for select genotypes. All line graphs have the same y‐axis scale and are shown for increased resolution in genotypes with low siRNA levels.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . A A representative microRNA , miR158B . location of miR158B from this locus is shown in orange .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.A A representative microRNA, miR158B. The location of miR158B from this locus is shown in orange.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . B SimpleHat2 non ‐ autonomous TE targeted by Pol IV ‐ RdDM . C RDR6 ‐ RdDM target locus TAS3a .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.B The SimpleHat2 non‐autonomous TE targeted by Pol IV‐RdDM.C The RDR6‐RdDM target locus TAS3a.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . D Athila6A consensus element in wt Col epigenome . E same Athila6A consensus element as in ( D ) upon transcriptional activation in ddm1 epigenome .

AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.D The Athila6A consensus element in the wt Col epigenome.E The same Athila6A consensus element as in (D) upon transcriptional activation in the ddm1 epigenome.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . F total small RNAs produced in wt Col TE ‐ silenced epigenome . G total small RNAs in ddm1 mutants .

AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.F The total small RNAs produced in the wt Col TE‐silenced epigenome.G The total small RNAs in ddm1 mutants.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . H Bisulfite sequencing of different regions of Athila6A element . Error bars represent Wilson score interval 95 % confidence limits . Differences in methylation status were analyzed with a two ‐ unpaired Student ' s t ‐ .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.H Bisulfite sequencing of different regions of the Athila6A element. Error bars represent Wilson score interval 95% confidence limits. Differences in the methylation status were analyzed with a two‐tailed unpaired Student's t‐test.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . I A second biological replicate of FLAG ‐ AGO6 ‐ IP no ‐ antigen control followed by deep sequencing shown for Athila6A consensus element in ddm1 mutants . transcriptional start sites of LTR TSS ‐ IR ‐ driven transcripts are shown .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.I A second biological replicate of the FLAG‐AGO6‐IP and no‐antigen control followed by deep sequencing shown for the Athila6A consensus element in ddm1 mutants. The transcriptional start sites of the LTR TSS‐ and IR‐driven transcripts are shown.


AGO6 relative enrichment of small RNAs ( solid lines ) total small RNAs ( dashed lines ) are shown for each small class ( 21 , 22 24nt ) at four genomic loci . J of first base of AGO6 ‐ enriched siRNAs from wt Col TE ‐ silenced epigenome ( left ) ddm1 TE ‐ transcriptionally active epigenome ( right ) .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.J Analysis of the first base of AGO6‐enriched siRNAs from the wt Col TE‐silenced epigenome (left) and the ddm1 TE‐transcriptionally active epigenome (right).


B H3Ac control ChIP from same used in AGO6 ‐ IP for At1g08200 constitutively expressed gene Athila6A TSS . Enrichment of H3Ac at Athila6A TSS is increased upon transcriptional activation in ddm1 mutants , while enrichment of H3Ac at gene At1g08200 is consistently high in both wt Col ddm1 . Data are represented as mean ± SD of six biological replicates for each genotype .

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B H3Ac control ChIP from the same chromatin used in the AGO6‐IP for the At1g08200 constitutively expressed gene and the Athila6A TSS. Enrichment of H3Ac at the Athila6A TSS is increased upon transcriptional activation in ddm1 mutants, while enrichment of H3Ac at the gene At1g08200 is consistently high in both wt Col and ddm1. Data are represented as mean ± SD of six biological replicates for each genotype.


C - E AGO6 ‐ ChIP qPCR of ( C ) gene At1g08200 Pol IV ‐ RdDM target SimpleHat2 , ( D ) Athila6A TSS in wt Col TE ‐ silenced epigenomes ( left ) ddm1 TE ‐ active epigenomes ( right ) ( E ) TAS3a . ChIP signals in ( D ) are normalized to ago6 or ddm1 ago6 no ‐ antigen controls . ChIP signals in ( C ) ( E ) are normalized to ago6 no ‐ antigen control . In ( C - E ) , each biological replicate data point is shown as a gray dot . data are represented as mean ± SD . For multiple statistical , one ‐ way ANOVA was performed using Tukey ' s multiple comparison .

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C-E AGO6‐ChIP qPCR analysis of (C) the gene At1g08200 and the Pol IV‐RdDM target SimpleHat2, (D) the Athila6A TSS in wt Col TE‐silenced epigenomes (left) and ddm1 TE‐active epigenomes (right) and (E) TAS3a. ChIP signals in (D) are normalized to the ago6 or ddm1 ago6 no‐antigen controls. ChIP signals in (C) and (E) are normalized to the ago6 no‐antigen control. In (C-E), each biological replicate data point is shown as a gray dot. The data are represented as mean ± SD. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test.


F ChIP qPCR of FLAG ‐ tagged POL V in wt Col ddm1 . ChIP signals are normalized to wt Col no ‐ antigen control .

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F ChIP qPCR analysis of the FLAG‐tagged POL V protein in wt Col and ddm1. ChIP signals are normalized to the wt Col no‐antigen control.


A AGO6 enrichment depletion of 21 - 22nt siRNAs for entire genome in 100 ‐ bp tiles for both wt Col ddm1 . Tiles were categorized by annotation feature .

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A AGO6 enrichment and depletion of 21-22nt siRNAs for the entire genome in 100‐bp tiles for both wt Col and ddm1. Tiles were categorized by annotation feature.


B Of tiles in ( A ) in both wt Col ddm1 , we determined number of tiles with ≥ 2 ‐ fold CHH methylation dependent on RDR6 .

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B Of the tiles in (A) in both wt Col and ddm1, we determined the number of tiles with ≥ 2‐fold CHH methylation dependent on RDR6.


C Distribution of AGO6 enrichment across TEs ( solid lines ) genes ( dashed lines ) for different sizes of small RNAs in either wt Col TE ‐ silenced epigenome or ddm1 TE ‐ transcriptionally active epigenome . Dashed lines have same color codes as solid lines . ' Start ' ' Stop ' represent edges of TE transcriptional start stop sites of genes .

C Distribution of AGO6 enrichment across TEs (solid lines) and genes (dashed lines) for different sizes of small RNAs in either the wt Col TE‐silenced epigenome or the ddm1 TE‐transcriptionally active epigenome. Dashed lines have the same color codes as the solid lines. 'Start' and 'Stop' represent the edges of the TE and the transcriptional start and stop sites of the genes.


D - G Heatmaps of AGO6 enrichment of TE siRNA classes in wt Col ddm1 epigenomes . Analyzed were ( D ) position of TE on a chromosome TE , ( E ) copy number of TE , ( F ) TE type ( G ) TE family name . color of TE family name in ( G ) corresponds to TE type in ( F ) .

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D-G Heatmaps of AGO6 enrichment of TE siRNA size classes in wt Col and ddm1 epigenomes. Analyzed were (D) the position of the TE on a chromosome and TE size, (E) the copy number of the TE, (F) the TE type and (G) the TE family name. The color of the TE family name in (G) corresponds to the TE type in (F).


H Bisulfite sequencing of TE targets . Error bars represent Wilson score interval 95 % confidence limits .

H Bisulfite sequencing of TE targets. Error bars represent Wilson score interval 95% confidence limits.


A Bisulfite sequencing of Athila6A TSS in juvenile leaf tissue .

A Bisulfite sequencing of the Athila6A TSS in juvenile leaf tissue.


B qRT - PCR of Athila6A gag / pol steady ‐ state mRNA transcript in inflorescence leaf tissue .

B qRT-PCR analysis of Athila6A gag/pol steady‐state mRNA transcript levels in inflorescence and leaf tissue.


C qRT - PCR of AGO6 AGO4 mRNA transcript in inflorescence juvenile leaf tissue .

C qRT-PCR analysis of AGO6 and AGO4 mRNA transcript levels in inflorescence and juvenile leaf tissue.


D Western blot of wt Col leaf inflorescence . arrow denotes AGO6 ; higher band is a previously observed cross ‐ reactive band ( see also Supplementary Fig S3A ) .

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D Western blot analysis of wt Col leaf and inflorescence. The arrow denotes the AGO6 protein; the higher band is a previously observed cross‐reactive band (see also Supplementary Fig S3A).


E , F Side view of an inflorescence with ( E ) or without ( F ) AGO6 ‐ GFP transgene . G , H Top ‐ down view of an inflorescence with ( G ) or without ( H ) AGO6 ‐ GFP transgene .

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E, F Side view of an inflorescence with (E) or without (F) the AGO6‐GFP transgene.G, H Top‐down view of an inflorescence with (G) or without (H) the AGO6‐GFP transgene.


J fractionation of inflorescence . cytoplasm ‐ enriched represents 2 . 5 % of total ‐ enriched . H3Ac is a marker , while PEPC is a .

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J Nuclear and cytoplasmic fractionation of inflorescence protein. The cytoplasm‐enriched protein represents 2.5% of the total nuclear‐enriched protein. H3Ac is a nuclear protein marker, while PEPC is a cytoplasmic protein.


K CHH methylation of RDR6 ‐ RdDM targets TAS3a Athila6A TSS in whole ‐ inflorescence ‐ tissue samples compared to dissected young buds corresponding to floral stages 6 - 8 .

K CHH methylation levels of the RDR6‐RdDM targets TAS3a and Athila6A TSS in whole‐inflorescence‐tissue samples compared to dissected young buds corresponding to floral stages 6-8.


( A ) Pearson correlation between ZEB1 MITF mRNA expression in 61 melanomacell lines available through CCLE .

(A) Pearson correlation between ZEB1 and MITF mRNA expression in 61 melanomacell lines available through the CCLE.


( B ) ZEB1 , ZEB2 , TWIST1 MITF expression in a panel of BRAFV600 - mutated melanomacells assessed by Western blot . GLO C - 09 . 10cells are - derived short - term cultures . Actin was used as a loading control .

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(B) ZEB1, ZEB2, TWIST1 and MITF expression in a panel of BRAFV600-mutated melanomacells assessed by Western blot. GLO and C-09.10cells are patient-derived short-term cultures. Actin was used as a loading control.


( C ) Quantitative PCR analyses of ZEB1 , ZEB2 , TWIST1and MITF in same panel of lines . mRNA expression are represented relatively to C - 09 . 10 , in which were arbitrarily fixed at 1 ( n = 3 , mean ± SD ) . dotted separates ZEB1high ( left ) ZEB1low ( right ) lines .

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(C) Quantitative PCR analyses of ZEB1, ZEB2, TWIST1and MITF in the same panel of cell lines. mRNA expression levels are represented relatively to C-09.10 cells, in which the levels were arbitrarily fixed at 1 (n=3, mean ± SD). The dotted line separates ZEB1high (left) and ZEB1low (right) cell lines.


( D ) Tukey box plot of ZEB1 , ZEB2 , TWIST1 , MITF mRNA expression according to IC50 of drug ( µM ) administered ( BRAFi / MEKi ) , in melanoma lines from CCLE ( n = 28 ) ( Student ' s t - ) . High ZEB1 , low ZEB2 , low MITF expression were correlated with BRAFi ( PLX4720 ) MEKi ( AZD6244 ) resistance . PLX4720 is an analog of PLX4032 .

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(D) Tukey box plot of ZEB1, ZEB2, TWIST1, and MITF mRNA expression according to the IC50 of the drug (µM) administered (BRAFi/MEKi), in melanoma cell lines from the CCLE (n=28) (Student's t-Test). High ZEB1, low ZEB2, and low MITF expression levels were correlated with BRAFi (PLX4720) and MEKi (AZD6244) resistance. PLX4720 is an analog of PLX4032.


( E ) IC50 values of PLX4032 ( µM ) in panel of BRAFV600 melanoma as determined by ATP ( n = 3 , mean ± SD ) . For SKMEL24 WM793 , IC50 was > 8µM .

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(E) IC50 values of PLX4032 (µM) in the panel of BRAFV600 melanoma cells as determined by ATP assay (n=3, mean ± SD). For SKMEL24 and WM793, IC50 was >8µM.


( A ) Pearson correlation between ZEB1 MITF mRNA expression in 467 melanomatumors from TCGA data set .

(A) Pearson correlation between ZEB1 and MITF mRNA expression levels in 467 melanomatumors from TCGA data set.


( B ) Representative pictures of ZEB1 MITF immunostaining in melanomas . Scale bar = 40 µm . aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype .

(B) Representative pictures of ZEB1 and MITF immunostaining in primary melanomas. Scale bar = 40 µm. The aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype.


( C ) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from , classified in ZEB1 high , int low subgroups , on of ZEB1 on of positive for ZEB1 . Scale bar = 80 µm . ( D ) Pie charts representing distribution of ZEB1 alone ( upper part ) , or ZEB1 TWIST1 ( lower part ) immunohistochemistry in tumors according to their initial response to vemurafenib + / - cobimetinib treatment . ZEB1 + / - TWIST1 are higher in MAPKi resistant melanomas ( initial non - responders ) compared to tumors that initially respond to treatment ( n = 70 , Fisher ' s exact ) .

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(C) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from patients, classified in ZEB1 high, int and low subgroups, based on the intensity of ZEB1 staining and on the percentage of cells positive for ZEB1. Scale bar = 80 µm. (D) Pie charts representing the distribution of ZEB1 alone (upper part), or ZEB1 and TWIST1 (lower part) immunohistochemistry staining in tumors according to their initial response to vemurafenib +/- cobimetinib treatment. ZEB1 +/- TWIST1 levels are higher in MAPKi primary resistant melanomas (initial non-responders) compared to tumors that initially respond to treatment (n=70, Fisher's exact test).


( E ) Representative pictures of ZEB1 MITF immunostainings , before after vemurafenib treatment , in from 1 , exhibiting resistance to BRAFi . Scale bar = 80 µm . Right : Magnification of MITFhigh MITFlow clones in resistant under treatment . Arrows indicate endothelial stromal that also show positive for ZEB1 , besides .

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(E) Representative pictures of ZEB1 and MITF immunostainings, before and after vemurafenib treatment, in the tumor from patient 1, exhibiting primary resistance to BRAFi. Scale bar = 80 µm. Right: Magnification of MITFhigh and MITFlow clones in the resistant tumor under treatment. Arrows indicate endothelial and stromal cells that also show positive staining for ZEB1, besides tumor cells.


( A ) PLX4032 IC50 ( µM ) of sensitive A375 SKMEL5 resistant ( A375 - R , SKMEL5 - R ) lines , as well as of GOKA ESPcells , two BRAFi - resistant - derived short - term cultures , as determined by ATP ( n = 3 , mean ± SD ) . For ESP , IC50 was > 8µM .

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(A) PLX4032 IC50 (µM) of sensitive A375 and SKMEL5 and resistant (A375-R, SKMEL5-R) cell lines, as well as of GOKA and ESPcells, two BRAFi-resistant patient-derived short-term cultures, as determined by ATP assay (n=3, mean ± SD). For ESP, IC50 was >8µM.


( B ) Western blot analyses of ZEB1 , TWIST1 FRA1 in A375 - R SKMEL5 - Rversus parental naive , in GOKA ESP . GAPDH was used as a loading control .

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(B) Western blot analyses of ZEB1, TWIST1 and FRA1 in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. GAPDH was used as a loading control.


( C ) Quantitative PCR analyses of ZEB1and MITF in A375 - R SKMEL5 - Rversus parental naive , in GOKA ESP . mRNA expression are represented as arbitrary units ( a . u ) . Statistical difference relative to sensitive A375 is shown ( n = 3 , mean ± SD , Student ' s t - ) .

(C) Quantitative PCR analyses of ZEB1and MITF in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. mRNA expression levels are represented as arbitrary units (a.u). Statistical difference relative to sensitive A375 cells is shown (n=3, mean ± SD, Student's t-test).


( D ) Representative pictures of ZEB1 MITF immunostainings in tumors from 2 , 3 4 , before after vemurafenib treatment . Scale bar = 40 µm . For ZEB1 in 4 , inset shows a magnification . Arrows point at stromal ( s ) . other positive for ZEB1 are .

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(D) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2, 3 and 4, before and after vemurafenib treatment. Scale bar = 40 µm. For ZEB1 staining in patient 4, the inset shows a magnification. Arrows point at stromal cells (s). All other cells positive for ZEB1 are tumor cells.


( A ) A375 were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 p75 . GAPDH was used as a loading control .

(A) A375 cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control.


( B ) Quantitative PCR analyses of MITF , p75 , ABCB5 JARID1B upon ZEB1 expression . mRNA expression are represented relatively to control , in which were fixed at 1 ( mean ± SD , n = 3 , Student ' s t - ) .

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(B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 expression. mRNA expression levels are represented relatively to control cells, in which the levels were fixed at 1 (mean ± SD, n=3, Student's t-test).


( C ) Soft agar colony formation upon ZEB1 expression . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) .

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(C) Soft agar colony formation assay upon ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( D ) 2 x 106 Control or ZEB1 - overexpressing A375 were injected subcutaneously into nude mice . mean volume for 5 mice is represented ± ( Student ' s t - ) .

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(D) 2 x 106 Control or ZEB1-overexpressing A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented ± SEM (Student's t-test).


( E ) Western blot analyses of ZEB1 , MITF , P - ERK , TWIST1 in control or ZEB1 - expressing + / - 150 nM PLX4032 treatment for 24 h . GAPDH was used as a loading control .

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(E) Western blot analyses of ZEB1, MITF, P-ERK, and TWIST1 levels in control or ZEB1-expressing cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.


( F ) FACS analyses of p75 surface expression upon ZEB1 overexpression , after 10 days treatment with or without 150 nM PLX4032 . Bar chart representing mean of p75 - high , int low from 2 independent experiments ( Fisher ' s Exact ) .

(F) FACS analyses of p75 cell surface expression upon ZEB1 overexpression, after 10 days treatment with or without 150 nM PLX4032. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's Exact Test).


( G ) Control or ZEB1 - overexpressing A375 were transfected with control or p75 - siRNA . p75 MITF expression were was analyzed by quantitative PCR after 48 h . mRNA expression are represented relatively to control transfected with siRNA Control , in which were fixed at 1 ( mean ± SD , n = 2 ) .

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(G) Control or ZEB1-overexpressing A375 cells were transfected with control or p75-siRNA. p75 and MITF expression levels were was analyzed by quantitative PCR after 48 h. mRNA expression levels are represented relatively to control cells transfected with siRNA Control, in which the levels were fixed at 1 (mean ± SD, n=2).


( H ) Clonogenic + / - PLX4032 ( 150 nM ) , + / - GDC - 0973 ( 5 nM ) treatment for 10 days . graphs represent mean number of colonies ( ± SD ) in 3 independent experiments ( Student ' s t - ) . ( I ) Number of of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1 - expressing ( n = 3 , Student ' s t - ) .

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(H) Clonogenic assay +/- PLX4032 (150 nM), +/- GDC-0973 (5 nM) treatment for 10 days. The graphs represent the mean number of colonies (± SD) in 3 independent experiments (Student's t-test). (I) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1-expressing cells (n=3, Student's t-test).


( A ) C - 09 . 10 short - term culture were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 p75 . GAPDH was used as a loading control .

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(A) C-09.10 short-term culture cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control.


( B ) Quantitative PCR analyses of MITF p75 upon ZEB1 expression . mRNA expression are represented relatively to control ( mean ± SD , n = 3 , Student ' s t - ) .

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(B) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).


( C ) Soft agar colony formation following ZEB1 expression . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) .

(C) Soft agar colony formation assay following ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( D ) FACS analyses of p75 surface expression upon ZEB1 overexpression . Bar chart representing mean of p75 - high , int , low negative from 2 independent experiments ( Fisher ' s exact ) .

(D) FACS analyses of p75 cell surface expression upon ZEB1 overexpression. Bar chart representing the mean percentage of p75-high, int, low and negative cells from 2 independent experiments (Fisher's exact test).


( E ) Clonogenic + / - PLX4032 ( 100 nM ) treatment for 10 days . graph represents mean number of colonies in 3 independent experiments ( Student ' s t - ) .

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(E) Clonogenic assay +/- PLX4032 (100 nM) treatment for 10 days. The graph represents the mean number of colonies in 3 independent experiments (Student's t-test).


( A ) A375 were infected with retroviruses expressing a control or ZEB1 - shRNA . Western blot analyses of ZEB1 p75 upon ZEB1 knock - down . GAPDH was used as a loading control . High low exposures ( exp ) for p75 are shown .

(A) A375 cells were infected with retroviruses expressing a control or ZEB1-shRNA. Western blot analyses of ZEB1 and p75 upon ZEB1 knock-down. GAPDH was used as a loading control. High and low exposures (exp) for p75 are shown.


( B ) Quantitative PCR analyses of MITF , p75 , ABCB5 JARID1B upon ZEB1 knock - down . mRNA expression are represented relatively to shRNA control ( mean ± SD , n = 3 , Student ' s t - ) .

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(B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 knock-down. mRNA expression levels are represented relatively to shRNA control cells (mean ± SD, n=3, Student's t-test).


( C ) FACS analyses of p75 expression upon ZEB1 knock - down . Bar chart representing mean of p75 - high , int low from 2 independent experiments ( Fisher ' s exact ) .

(C) FACS analyses of p75 expression upon ZEB1 knock-down. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's exact test).


( D ) Soft agar colony formation upon ZEB1 knock - down . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) .

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(D) Soft agar colony formation assay upon ZEB1 knock-down. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( E ) 2 x 106 Control or ZEB1 - shRNA A375 were injected subcutaneously into nude mice . mean volume for 5 mice is represented ( ± ) . ( Student ' s t - ) .

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(E) 2 x 106 Control or ZEB1-shRNA A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( F ) A375 were infected with an IPTG - inducible ZEB1 - shRNA . Left panel : Western blot analyses of ZEB1 expression ± IPTG ( 100µM ) treatment for 6 days . Actin was used as a loading control . Right panel : 2 x 106 IPTG - inducible shRNA - Control or shRNA - ZEB1A375 were injected subcutaneously into nude mice . When reached 5 mm in diameter , ZEB1 expression was silenced by adding IPTG ( 10 mM ) into drinking water for 20 days . mean volume for 5 mice is represented ( ± ) . ( Student ' s t - ) .

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(F) A375 cells were infected with an IPTG-inducible ZEB1-shRNA. Left panel: Western blot analyses of ZEB1 expression ± IPTG (100µM) treatment for 6 days. Actin was used as a loading control. Right panel: 2 x 106 IPTG-inducible shRNA-Control or shRNA-ZEB1A375 cells were injected subcutaneously into nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by adding IPTG (10 mM) into the drinking water for 20 days. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( G ) SKMEL5 expressing an IPTG - inducible ZEB1 - shRNA were treated with IPTG ( 100µM ) for 10 days ( + IPTG ) , then IPTG was removed ( - IPTG ) ZEB1 , MITF p75 expression were analyzed by Western blot / or quantitative PCR analyses . GAPDH was used as a loading control , mRNA expression are represented relatively to untreated , in which were fixed at 1 ( mean ± SD , n = 2 ) .

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(G) SKMEL5 cells expressing an IPTG-inducible ZEB1-shRNA were treated with IPTG (100µM) for 10 days (+IPTG), then IPTG was removed (-IPTG) and ZEB1, MITF and p75 expression levels were analyzed by Western blot and/or quantitative PCR analyses. GAPDH was used as a protein loading control, mRNA expression levels are represented relatively to untreated cells, in which the levels were fixed at 1 (mean ± SD, n=2).


( A ) Western blot analyses of ZEB1 P - ERK in shRNA - control or shRNA - ZEB1 - expressing A375 + / - 150 nM PLX4032 treatment for 24 h . GAPDH was used as a loading control .

(A) Western blot analyses of ZEB1 and P-ERK in shRNA-control or shRNA-ZEB1-expressing A375 cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.


( B ) Soft agar colony formation in A375 in presence or absence of PLX4032 ( 150 nM ) . Scale bar = 100 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - ) . ( C ) Number of of chronic exposure to PLX4032 before emergence of resistance in control or shRNA - ZEB1 - expressing ( n = 3 , Student ' s t - ) .

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(B) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 (150 nM). Scale bar = 100 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (C) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA-ZEB1-expressing cells (n=3, Student's t-test).


( D ) RPMI7951 , A375 - R SKMEL5 - R were infected with a retrovirus encoding a constitutive shRNA - ZEB1 . Short - term cultures of GOKA ESP , derived from vemurafenib - resistant , were infected with a lentivirus encoding an IPTG - inducible shRNA - ZEB1 . Western blot analyses showing efficient ZEB1 knock - down in different models , ± IPTG ( 200µM ) , ± PLX4032 ( 3µM ) as indicated . Induction of was assessed by PARP cleavage . GAPDH or actin were used as loading control .

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(D) RPMI7951, A375-R and SKMEL5-R were infected with a retrovirus encoding a constitutive shRNA-ZEB1. Short-term cultures of GOKA and ESP cells, derived from vemurafenib-resistant patients, were infected with a lentivirus encoding an IPTG-inducible shRNA-ZEB1. Western blot analyses showing efficient ZEB1 knock-down in the different models, ±IPTG (200µM), ±PLX4032 (3µM) as indicated. Induction of cell death was assessed by PARP cleavage. GAPDH or actin were used as loading control.


( E ) Clonogenic assays in presence of 3 µM PLX4032 , with or without IPTG ( 200 µM ) as indicated . graphs represent mean number of colonies in 3 independent experiments ( Student ' s t - ) .

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(E) Clonogenic assays in the presence of 3 µM PLX4032, and with or without IPTG (200 µM) as indicated. The graphs represent the mean number of colonies in 3 independent experiments (Student's t-test).


( F ) Western blot analyses of ZEB1 , p75 quantitative PCR analyses of MITF in shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant - derived short - term culture . Actin was used as a loading control . mRNA expression are represented relatively to control ( mean ± SD , n = 3 , Student ' s t - ) .

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(F) Western blot analyses of ZEB1, p75 and quantitative PCR analyses of MITF in shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant patient-derived short-term culture cells. Actin was used as a loading control. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).


( G ) 2 , 5 x 106 shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant were injected subcutaneously in nude mice . When reached 5 mm in diameter , ZEB1 expression was silenced by providing mice with IPTG ( 10 mM ) in their drinking water orally administering vemurafenib ( 50 mg / kg ) daily for 4 . mean volume for 5 mice is represented ( ± ) . ( Student ' s t - ) .

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(G) 2,5 x 106 shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant cells were injected subcutaneously in nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by providing mice with IPTG (10 mM) in their drinking water and orally administering vemurafenib (50 mg/kg) daily for 4 weeks. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).


( H ) Upper part : Western blot analyses of ZEB1 in shRNA - Control ( 1 to 4 ) or shRNA - ZEB1 ( 5 to 8 ) ESPxenografttumors , showing efficient ZEB1 knock - down directly in tumors , after IPTG ± PLX4032 treatment in vivo . Lower part : Representative pictures of ZEB1 immunostaining in shRNA - Control or shRNA - ZEB1tumors , after IPTG treatment in vivo . Scale bar = 40µm .

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(H) Upper part: Western blot analyses of ZEB1 in shRNA-Control (1 to 4) or shRNA-ZEB1 (5 to 8) ESPxenografttumors, showing efficient ZEB1 knock-down directly in the tumors, after IPTG ±PLX4032 treatment in vivo. Lower part: Representative pictures of ZEB1 immunostaining in shRNA-Control or shRNA-ZEB1tumors, after IPTG treatment in vivo. Scale bar = 40µm.


A Frequency of MN leukemic expressing immature marker cKit mature myeloid marker Ly6G in peripheral blood following 5 days of treatment ( n = 9 - 10 mice / group ; mice with < 2 % burden in any condition were censored from ) . Data information : data in A are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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A Frequency of MN leukemic cells expressing the immature marker cKit and mature myeloid marker Ly6G in the peripheral blood following 5 days of treatment (n = 9-10 mice/group; mice with < 2% tumor burden in any condition were censored from the analysis). Data information: data in A are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


B Absolute number of MN in peripheral blood quantified by flow cytometry . Grey bars denote treatment ( n = 11 - 12 mice / group ) . Data information : Dox - doxycycline .

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B Absolute number of MN cells in the peripheral blood quantified by flow cytometry. Grey bars denote treatment (n = 11-12 mice/group). Data information: Dox - doxycycline.


C Kaplan - Meier survival curve of leukemic mice ( n = 11 - 12 mice / group , median survival is 16 . 5 for vehicle not reached for AG636 doxycycline , P < 0 . 0001 by Log - rank ) . Data information : Dox - doxycycline .

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C Kaplan-Meier survival curve of leukemic mice (n = 11-12 mice/group, median survival is 16.5 for vehicle and not reached for AG636 and doxycycline, P < 0.0001 by Log-rank test). Data information: Dox - doxycycline.


D Representative FACS plots showing differentiation induced by AG636 doxycycline . Data information : Dox - doxycycline .

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D Representative FACS plots showing differentiation induced by AG636 and doxycycline. Data information: Dox - doxycycline.


E Spleen weights of MN - bearing mice ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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E Spleen weights of MN tumor-bearing mice (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


F Absolute number of MN in bone marrow ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

F Absolute number of MN cells in the bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


G Frequency ( as of MN ) of LSCs ( CD11blowcKithighFcgR + ) differentiated ( CD182 + Ly6G + ) in bone marrow ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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G Frequency (as percentage of all MN cells) of LSCs (CD11blowcKithighFcgR+) and differentiated cells (CD182+Ly6G+) in the bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


H May - Grunwald - Giemsa - stained cytospins of sorted MN showing myeloid differentiation . Data information : Dox - doxycycline .

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H May-Grunwald-Giemsa-stained cytospins of sorted MN cells showing myeloid differentiation. Data information: Dox - doxycycline.


I of functionally - define LSCs using a limiting dilution calculated by ELDA ( n = 4 - 8 mice / group ) .

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I Quantification of functionally-define LSCs using a limiting dilution assay calculated by ELDA (n = 4-8 mice/group).


A Spleen weights of RUNX1 - RUNX1T1 or I1DN bearing mice treated with AG636 or vehicle for 4 days . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 .

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A Spleen weights of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


B - C Absolute number of leukemic in bone marrow ( B ) spleens ( C ) of RUNX1 - RUNX1T1 or I1DN bearing mice treated with AG636 or vehicle for 4 days . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 .

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B-C Absolute number of leukemic cells in the bone marrow (B) and spleens (C) of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


D - E Frequency of leukemic expressing immature marker cKit mature myeloid marker CD11b in bone marrow spleens of RUNX1 - RUNX1T1 ( D ) or I1DN ( E ) bearing mice treated with AG636 or vehicle for 4 days ( n = 6 mice / group ) . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - Student ' s unpaired t - . * P < 0 . 05 , * * P < 0 . 01 .

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D-E Frequency of leukemic cells expressing the immature marker cKit and mature myeloid marker CD11b in the bone marrow and spleens of RUNX1-RUNX1T1 (D) or I1DN (E) tumor bearing mice treated with AG636 or vehicle for 4 days (n = 6 mice/group). Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


Various bone marrow populations were quantified by flow cytometry in mice treated with AG636 or vehicle for 1 or 4 days ( acute treatment ) or for 4 cycles followed by 4 off treatment ( post - recovery ) . Data information : n = 4 mice / group for acute treatment , n = 5 - 6 mice for prolonged treatment recovery ; data are presented as mean ± s . d . ; P values were calculated using a two - Student ' s unpaired t - ; only comparisons that meet threshold of P < 0 . 05 are shown . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 .

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Various bone marrow populations were quantified by flow cytometry in mice treated with AG636 or vehicle for 1 or 4 days (acute treatment) or for 4 cycles followed by 4 weeks off treatment (post-recovery). Data information: n = 4 mice/group for acute treatment, n = 5-6 mice for prolonged treatment and recovery; data are presented as mean ± s.d.; P values were calculated using a two-tailed Student's unpaired t-test; only comparisons that meet the threshold of P < 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001


E - H Peripheral blood red blood ( E ) , platelets ( F ) , myeloid ( G ) lymphoid ( H ) quantified in mice treated with AG636 or vehicle . Grey bars denote treatment .

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E-H Peripheral blood red blood cells (E), platelets (F), myeloid cells (G) and lymphoid cells (H) quantified in mice treated with AG636 or vehicle. Grey bars denote treatment.


sequencing performed on cKithighCD11blow MN sorted from AG636 , doxycycline or vehicle treated mice ( n = 3 mice / group ) . Gene set enrichment showing common differential enrichment of biological pathways in gene expression data from AG636 or doxycycline treated . Gene sets are from C2 : CGP Reactome sub - collections in MSigDB database ( B ) .

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RNA sequencing performed on cKithighCD11blow MN cells sorted from AG636, doxycycline or vehicle treated mice (n = 3 mice/group). Gene set enrichment analysis showing common and differential enrichment of biological pathways in gene expression data from AG636 or doxycycline treated animals. Gene sets are from C2:CGP and Reactome sub-collections in MSigDB database (B).


C sequencing performed on cKit + CD11b - RUNX1 - RUNX1T1 or I1DN sorted from mice treated with AG636 or vehicle for 1 day ( n = 3 mice / group ) . Bar code plots showing enrichment of selected pathways .

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C RNA sequencing performed on cKit+CD11b- RUNX1-RUNX1T1 or I1DN cells sorted from mice treated with AG636 or vehicle for 1 day (n = 3 mice/group). Bar code plots showing enrichment of selected pathways.


E - F qPCR showing down - regulation of genes encoding ribosomal in MN ( E ) human AML lines ( F ) treated for 24 hours with AG636 ( n = 3 biological replicates for each ) . Data information : data in E , F are presented as mean ± s . e . m . ; P values were calculated using a one - Student ' s unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ;

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E-F qPCR showing down-regulation of genes encoding ribosomal proteins in MN cells (E) and human AML cell lines (F) treated for 24 hours with AG636 (n = 3 biological replicates for each cell line). Data information: data in E, F are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;


G - H Polysome profiling of MN treated in vitro with AG636 or vehicle for 24h . Representative trace ( G ) of sub - polysome heavy polysome fractions ( H ) determined by measuring under curve ( AUC ) ( n = 4 biological replicates ) . Data information : data in are presented as mean ± s . e . m . ; P values were calculated using a one - Student ' s unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ;

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G-H Polysome profiling of MN cells treated in vitro with AG636 or vehicle for 24h. Representative trace (G) and quantification of sub-polysome and heavy polysome fractions (H) determined by measuring area under the curve (AUC) (n = 4 biological replicates). Data information: data in are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;


I Nascent synthesis quantified using AHA incorporation in MN or MOLM13 treated with AG636 for 24 hours or cycloheximide for 1 hour . that were cultured in absence of AHA served as a negative control . ( n = 3 biological replicates ) . Data information : data in I are presented as mean ± s . e . m . ; P values were calculated using a one - Student ' s unpaired t - ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; CHX - cycloheximide .

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I Nascent protein synthesis quantified using the AHA incorporation assay in MN or MOLM13 cells treated with AG636 for 24 hours or cycloheximide for 1 hour. Cells that were cultured in the absence of AHA served as a negative control. (n = 3 biological replicates). Data information: data in I are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; CHX - cycloheximide.