D . Zebrafish at one - stage were microinjected with plasmids as indicated , followed by extraction of embryonic at 96 - hpf stage . of CHOP , p - eIF2α , total eIF2α , Endou - Flag , its variants were detected using Western blot . GAPDH served as an internal control . E . HEK 293T were transfected with indicated plasmids followed by of of CHOP , p - eIF2α , total eIF2α , Endou - Flag , its variants using Western blot . α - tubulin served as an internal control . relative to each internal control are presented in each lane .

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

D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated, followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane.


D . Zebrafish at one - stage were microinjected with plasmids as indicated followed by extraction of embryonic at 96 - hpf stage . of CHOP , p - eIF2α , total eIF2α , Endou - Flag , its variants were detected using Western blot . GAPDH served as an internal control . E . HEK 293T were transfected with indicated plasmids followed by of of CHOP , p - eIF2α , total eIF2α , Endou - Flag , its variants using Western blot . Here , α - tubulin served as an internal control . relative to each internal control were presented in each lane .

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

D. Zebrafish embryos at one-cell stage were microinjected with plasmids as indicated followed by extraction of embryonic proteins at the 96-hpf stage. Protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants were detected using Western blot analysis. GAPDH served as an internal control. E. HEK 293T cells were transfected with indicated plasmids followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot . Here, α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.


B . HEK 293T were transfected with plasmids as indicated for 24 hr treated with DMSO , thapsigargin ( TH ) , ISRIB , or TH plus ISRIB for 2 hr followed by of level of CHOP , p - eIF2α , total eIF2α , Endou - Flag , its variants using Western blot . α - tubulin served as an internal control . relative to each internal control were presented at each lane .

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

B. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO, thapsigargin (TH), ISRIB, or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented at each lane.


D , E . MEF WT mutant S51A were transfected with plasmids as indicated for 24 hr treated with either DMSO or TH for 4 hr followed by of of CHOP , p - eIF2α , total eIF2α , GADD34 , Endouc - Flag using Western blot . GAPDH served as an internal control . relative to each internal control were presented at each lane .

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

D, E. MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, GADD34, and Endouc-Flag using Western blot. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane.


A . MEF wild - type ( WT ) were transfected with plasmids as indicated for 24 hr treated with either DMSO or Thapsigargin ( TH ) for 4 hr followed by of level of p - PKR , total PKR , Endouc - Flag . GAPDH served as an internal control . relative to each internal control were presented at each lane . results obtained from two trials are shown on left right .

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

A. MEF wild-type (WT) cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or Thapsigargin (TH) for 4 hr followed by analysis of the protein level of p-PKR, total PKR, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.


B . MEF WT were transfected with plasmids as indicated for 24 hr treated with either DMSO or TH for 4 hr followed by of of p - eIF2α , total eIF2α , CHOP , Endouc - Flag . GAPDH served as an internal control . relative to each internal control were presented at each lane . results obtained from two trials are shown on left right .

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

B. MEF WT cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of p-eIF2α, total eIF2α, CHOP, and Endouc-Flag. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. The results obtained from the two trials are shown on the left and right.


D , E . cleavage activity of Endouc was studied using materials presented at each lane . EndoucK242A : Endouc mutant ; Extract : incubated in extracts ; Buf : reaction buffer . probe without adding reaction buffer served as a negative control . predicted cleavage sites ( U ) were mutated to generate mutant huORFchop transcript ( unlabelled huORF - mt ) labelled by biotin ( biotin - huORF - mt ) . Unlabelled competitor was added in reactions at five times higher than that of biotinylated probe .

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

D, E. The cleavage activity of Endouc was studied using materials presented at each lane. EndoucK242A: Endouc mutant; Extract: incubated in cell extracts; and Buf: reaction buffer. RNA probe without adding reaction buffer served as a negative control. The predicted cleavage sites (U) were mutated to generate mutant huORFchop transcript (unlabelled huORF-mt) and labelled by biotin (biotin-huORF-mt). Unlabelled competitor RNA was added in reactions at five times higher than that of the biotinylated probe.


A . HEK 293T were transfected with pCS2 vector ( black ) , pCS2 - Endouc ( red ) , pCS2 - EndoucK242A ( green ) for 24 hr subjected to either DMSO ( control ) or thapsigargin ( TH ; stress ) treatment for 1 hr . lysates of transfected were applied to polysome profile ( PPA ) . sucrose gradient from top to bottom was shown from left to right , respectively . sedimentation of 40S , 60S , 80S , polysomes was indicated . Lysate from control , Endouc - expressing , EndoucK242A - expressing were applied to PPA . resulting fractions were TCA - precipitated , analysed by SDS - PAGE , subjected to Western blot using antibodies against Flag S6 .

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

A. HEK 293T cells were transfected with pCS2 vector (black), pCS2-Endouc (red), and pCS2-EndoucK242A (green) for 24 hr and subjected to either DMSO (control) or thapsigargin (TH; stress) treatment for 1 hr. The lysates of transfected cells were applied to polysome profile analysis (PPA). The sucrose gradient from top to bottom was shown from left to right, respectively. The sedimentation of 40S, 60S, 80S, and polysomes was indicated. Lysate from control, Endouc-expressing, and EndoucK242A-expressing cells were applied to PPA. The resulting fractions were TCA-precipitated, analysed by SDS-PAGE, and subjected to Western blot using antibodies against Flag and S6.


B . luc activity of Fluc ( solid column ) versus Rluc ( blank column ) in HEK 293T transfected with bicistronic mRNAs shown in Figures A was presented as an increase in fold over that obtained from control group of RF which was normalized as 1 . These data suggested that change of Fluc / Rluc shown in Figures A was entirely dependent on change of Fluc activity . data were averaged from three independent experiments presented as mean ± S . D . ( n = 3 ) . Student ' s t was used to determine significant differences between each group ( * * : P < 0 . 005 ) .

O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE I-CELL_LINE 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 O B-GENEPROD O B-ORGANISM 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 O O O O O O O O O O O O O O O O O O

B. The luc activity of Fluc (solid column) versus Rluc (blank column) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures A was presented as an increase in fold over that obtained from control group of RF which was normalized as 1. These data suggested that the change of Fluc/Rluc ratio shown in Figures A was entirely dependent on the change of Fluc activity. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (**: P<0.005).


D . luc activity of Fluc ( solid column ) versus Rluc ( blank column ) in HEK 293T transfected with bicistronic mRNAs shown in Figures C was presented as an increase in fold over that obtained from control group of hpRF which was normalized as 1 . These data suggested that change of Fluc / Rluc shown in Figures C was entirely dependent on change of Fluc activity . data were averaged from three independent experiments presented as mean ± S . D . ( n = 3 ) . Student ' s t was used to determine significant differences between each group ( * * * : P < 0 . 001 ) .

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

D. The luc activity of Fluc (solid column) versus Rluc (blank column) in HEK 293T cells transfected with bicistronic mRNAs shown in Figures C was presented as an increase in fold over that obtained from control group of hpRF which was normalized as 1. These data suggested that the change of Fluc/Rluc ratio shown in Figures C was entirely dependent on the change of Fluc activity. The data were averaged from three independent experiments and presented as mean ± S.D. (n = 3). Student's t test was used to determine significant differences between each group (***: P <0.001).


A - F . Schematic representation of capped ( m7G - capped ) A - capped ( non - functional capped ) monocistronic mRNAs with poly ( A ) - tail - encoded firefly luciferase ( Fluc ) fused upstream with ( A ) c - myc IRES , ( B ) no insertion , ( C ) non - specific 105 - nt luc ( Non105 ) , ( D ) huORFchop - 1 - 105 - nt , ( E ) huORFchop - 69 - 105 - nt , ( F ) huORFchop - 81 - 105 - nt as shown above each bar graph . transfection of each transcript equipped with m7G - capped or A - capped monocistronic mRNA in HEK 293T was compared . To normalize luc activities of capped A - capped Fluc transcripts , m7G - capped Renilla luc ( Rluc ) mRNA was used as an internal control . luc activity of transfected with Cap - monocistronic mRNA in each graph was set as 100 % .

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

A-F. Schematic representation of capped (m7G-capped) and A-capped (non-functional capped) monocistronic mRNAs with poly(A)-tail-encoded firefly luciferase (Fluc) and fused upstream with (A) c-myc IRES, (B) no insertion, (C) non-specific 105-nt luc RNA (Non105), (D) huORFchop-1-105-nt, (E) huORFchop-69-105-nt, and (F) huORFchop-81-105-nt as shown above each bar graph. The RNA transfection assay of each transcript equipped with m7G-capped or A-capped monocistronic mRNA in HEK 293T cells was compared. To normalize the luc activities of capped and A-capped Fluc transcripts, the m7G-capped Renilla luc (Rluc) mRNA was used as an internal control. The luc activity of cells transfected with Cap-monocistronic mRNA in each graph was set as 100%.


( b ) Representative electron micrograph of a viroplasm ( V ) formed in NSP5 - EGFP infected with rotaviruses 8 hours post infection . Inset - double - layered particles emerging from surface of a viroplasm . Scale bar = 300 nm .

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

(b) Representative electron micrograph of a viroplasm (V) formed in NSP5-EGFP cells infected with rotaviruses 8 hours post infection. Inset - double-layered particles emerging from the surface of a viroplasm. Scale bar = 300 nm.


( a ) recovery after photobleaching ( FRAP ) of EGFP - tagged replication factories after 4 HPI ( early infection ) 12 HPI ( late infection ) . Scale bar , 10 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY 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

(a) Fluorescence recovery after photobleaching (FRAP) of EGFP-tagged replication factories after 4 HPI (early infection) and 12 HPI (late infection). Scale bar, 10 μm.


( b ) Sensitivity of RV replication factories to aliphatic alcohols at 4 HPI . Left to right - 1 , 6 - hexanediol ( 1 , 6HD ) ; 1 , 3 - propylene diol ( 1 , 3PD ) ; 1 , 2 - propylene diol , or propylene glycol ( PG ) ; glycerol ( Gly ) ; polyethylene glycol 200 ( PEG200 ) . Top panels - before application bottom panels - 1 min after application of these compounds ( 4 . 5 % v / v ) . Scale bar , 30 µm .

O O O O O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 O O O O O O O O

(b) Sensitivity of RV replication factories to aliphatic alcohols at 4 HPI. Left to right -1,6-hexanediol (1,6HD); 1,3-propylene diol (1,3PD); 1,2-propylene diol, or propylene glycol (PG); glycerol (Gly); polyethylene glycol 200 (PEG200). Top panels - before application and bottom panels - 1 min after application of these compounds (4.5% v/v). Scale bar, 30 µm.


( a ) Recombinantly expressed , Atto647 - dye labelled NSP5 , Atto488 - dye labelled NSP2 unlabeled NSP5 samples were used for investigating their phase - separation properties . labelled samples were mixed with unlabeled NSP5 ( 1 : 10 molar ) to minimise effect of labelling on LLPS . Top panel , left to right : NSP5 - 647 / NSP2 - 488 droplets formed upon mixing of both ( 10 μM each ) . Both channels are shown , along with an image of both channels overlaid . 4 % ( v / v ) 1 , 6 - hexanediol ( 1 , 6HD ) dissolves these condensates . Scale bar , 10 µm . Middle panel , left to right : NSP5 - 647 ( 50 μM ) + poly - arginine ( polyArg , 5 μΜ ) ; NSP5 - polyArg condensate + 1 , 6 - HD ; NSP5 - polyArg condensates + 0 . 5 M NaCl ; NSP5 + R9 peptide ( Arg - 9 , 5 μΜ ) . Bottom row , left to right : NSP5 - 647 sample ( 35 μM ) alone ; after addition of 10 % v / v PEG - 20K ; NSP2 - 488 ( 25 μM ) alone ; after addition of 10 % v / v PEG - 20K .

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

(a) Recombinantly expressed, Atto647-dye labelled NSP5, Atto488-dye labelled NSP2 and unlabeled NSP5 protein samples were used for investigating their phase-separation properties. All labelled protein samples were mixed with the unlabeled NSP5 (1:10 molar ratio) to minimise the effect of labelling on LLPS. Top panel, left to right: NSP5-647/NSP2-488 droplets formed upon mixing of both proteins (10 μM each). Both channels are shown, along with an image of both channels overlaid. 4% (v/v) 1,6-hexanediol (1,6HD) dissolves these condensates. Scale bar, 10 µm. Middle panel, left to right: NSP5-647 (50 μM) + poly-arginine (polyArg, 5 μΜ); NSP5-polyArg condensate + 1,6-HD; NSP5-polyArg condensates + 0.5 M NaCl; NSP5 + R9 peptide (Arg-9, 5 μΜ). Bottom row, left to right: NSP5-647 sample (35 μM) alone; after addition of 10% v/v PEG-20K; NSP2-488 (25 μM) alone; after addition of 10% v/v PEG-20K.


Top panel , left to right : NSP5 - ∆ CTR labelled with Atto - 647 ( 25 μM ) incubated with unlabeled NSP2 ( 10 μΜ ) ; NSP5 - ∆ CTR in presence of 10 % v / v PEG - 20K ; with 5 μΜ of poly - arginine ( polyArg ) . Bottom panel : Atto - 647 - labelled NSP5 - ∆ CTR ( 5 μΜ ) incubated with unlabeled NSP5 ( 25 μΜ ) Atto - 488 labelled NSP2 ( 10 μΜ ) . NSP5 / NSP2 droplets containing labelled NSP2 ( green ) also contain NSP5 - ∆ CTR - Atto647 ( magenta ) , shown along with an image of both 488 / 647 channels overlaid . Scale bar , 10 µm .

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

Top panel, left to right: NSP5-∆CTR labelled with Atto-647 (25 μM) incubated with unlabeled NSP2 (10 μΜ); NSP5-∆CTR in the presence of 10% v/v PEG-20K; and with 5 μΜ of poly-arginine (polyArg). Bottom panel: Atto-647-labelled NSP5-∆CTR (5 μΜ) incubated with unlabeled NSP5 (25 μΜ) and Atto-488 labelled NSP2 (10 μΜ). NSP5/NSP2 droplets containing labelled NSP2 (green) also contain NSP5-∆CTR-Atto647 (magenta), shown along with an image of both 488/647 channels overlaid. Scale bar, 10 µm.


( a ) RV - infected MA - NSP5 - EGFP at 6 HPI . NSP5 - EGFP - tagged viral factories ( green ) are dissolved in presence of 4 . 7 % ( v / v ) propylene glycol ( PG , middle ) . Viral - condensates rapidly reform ( < 10 min ) after replacing PG - containing culture medium ( PG removed , right ) . Seg3 ( magenta ) Seg4 ( cyan ) transcripts are detected by smFISH , colocalising Seg3 Seg4 signals ( ) . Scale bars : 50 µm , zoomed - in regions : 10 µm .

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

(a) RV-infected MA-NSP5-EGFP cells at 6 HPI. NSP5-EGFP-tagged viral factories (green) are dissolved in the presence of 4.7% (v/v) propylene glycol (PG, middle). Viral RNA-protein condensates rapidly reform (<10 min) after replacing the PG-containing cell culture medium (PG removed, right). Seg3 (magenta) and Seg4 (cyan) transcripts are detected by smFISH, and colocalising Seg3 and Seg4 RNA signals (white). Scale bars: 50 µm, zoomed-in regions: 10 µm.


Effects of PG treatments on viral production . PG - containing medium ( 2 % or 4 . 7 % PG ) , or standard culture medium , were applied to RV - infected ( MOI of 10 ) 2 HPI ( 1 hour after absorption ) . ( e ) Effects of PG treatments on production at different infection points . 4 . 7 % ( v / v ) PG was applied between 2 - 7 HPI ( ' early infection ' ) , then diluted to 2 % between 7 - 11 HPI , or 4 . 7 % PG was applied between 7 - 11 HPI ( ' late infection ' ) , diluted prior to harvesting . Each group was compared to control group ( 10 h treatment with 2 % PG ) . Each group was compared to control group ( 10 h treatment with 2 % PG ) . Application of 4 . 7 % PG for 5 h significantly reduced replication ( P = 0 . 0016 ) between 2 - 7 HPI compared to control group . PG treatment between 7 - 12 HPI did not significantly reduce viral titre compared to control group ( P = 0 . 1141 ) . Infectious titres represent mean ± SD values estimated for four independent biological replicates . Statistical analyses were performed using a Kruskal - Wallis , followed by uncorrected Dunn ' s multiple comparisons ( * * P < 0 . 002 ) .

O O B-SMALL_MOLECULE O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE 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 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 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 O O O O O B-SMALL_MOLECULE O O O O O 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 B-SMALL_MOLECULE 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 O O O O O O O O O O O O

Effects of PG treatments on the viral production. PG-containing medium (2% or 4.7% PG), or standard cell culture medium, were applied to RV-infected cells (MOI of 10) 2 HPI (1 hour after virus absorption). (e) Effects of PG treatments on virus production at different infection points. 4.7% (v/v) PG was applied between 2-7 HPI ('early infection'), and then diluted to 2% between 7-11 HPI, or 4.7% PG was applied between 7-11 HPI ('late infection'), and diluted prior to harvesting the virus. Each group was compared to the control group (10 h treatment with 2% PG). Each group was compared to the control group (10 h treatment with 2% PG). Application of 4.7% PG for 5 h significantly reduced virus replication (P = 0.0016) between 2-7 HPI compared to the control group. PG treatment between 7-12 HPI did not significantly reduce the viral titre compared to the control group (P = 0.1141). Infectious titres represent mean±SD values estimated for four independent biological replicates. Statistical analyses were performed using a Kruskal-Wallis test, followed by uncorrected Dunn's multiple comparisons test (**P < 0.002).


( f , g ) 4 . 7 % PG treatment of RV - infected results in NSP5 dephosphorylation . ( f ) Western blot of RV - infected harvested at 7 12 HPI , treated with 2 % or 4 . 7 % PG at different infection time points indicated in figure . Multiple phosphorylated forms of NSP5 can be seen as higher MW bands . Treatment with 4 . 7 % PG reduces phosphorylation , which is not perturbed in presence of 2 % PG . Treatments administered between 2 - 7 7 - 12 hpi reduce NSP5 phosphorylation , albeit to a considerably lower degree compared to 2 - 12 hpi treatment . ( g ) Okadaic acid ( 5 μM ) applied to 4 . 7 % PG - treated infected blocks NSP5 dephosphorylation restoring its phosphorylation pattern .

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

(f, g) 4.7% PG treatment of RV-infected cells results in NSP5 dephosphorylation. (f) Western blot analysis of RV-infected cells harvested at 7 and 12 HPI, treated with 2% or 4.7% PG at different infection time points indicated in the figure. Multiple phosphorylated forms of NSP5 can be seen as higher MW bands. Treatment with 4.7% PG reduces phosphorylation, which is not perturbed in the presence of 2% PG. Treatments administered between 2-7 and 7-12 hpi reduce NSP5 phosphorylation, albeit to a considerably lower degree compared to the 2-12 hpi treatment. (g) Okadaic acid (5 μM) applied to 4.7% PG-treated infected cells blocks NSP5 dephosphorylation restoring its phosphorylation pattern.


( e ) 3D - PAINT of Seg3 foci in RV - infected at 4 6 HPI . Scale bars , 2 µm ( left ) 200 nm ( zoomed - in , right ) . ( f - g ) Distribution of calculated volumes sphericities of Seg3 - containing granules in RV - infected at 4 HPI ( N = 704 ) 6 hpi ( N = 698 ) , shown in ( e ) . Box plots represent 25th / 75th interquartile range , with whiskers representing 5th / 95th percentile values . Medians shown as central bands , means shown as squares . Symbol ' x ' denotes 1 % 99 % percentile values , min max values are shown as ' - ' . At 0 , 001 level , two distributions are significantly different between 4 6 hpi , assessed by two - sample Kolmogorov - Smirnov .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD 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 B-GENEPROD O O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3D DNA-PAINT analysis of Seg3 RNA foci in RV-infected cells at 4 and 6 HPI. Scale bars, 2 µm (left) and 200 nm (zoomed-in, right). (f-g) Distribution of calculated volumes and sphericities of the Seg3 RNA-containing granules in RV-infected cells at 4 HPI (N=704) and 6 hpi (N=698), shown in (e). Box plots represent the 25th/75th interquartile range, with whiskers representing the 5th/95th percentile values. Medians shown as central bands, and means shown as squares. Symbol 'x' denotes 1% and 99% percentile values, and min and max values are shown as '-'. At the 0,001 level, the two distributions are significantly different between 4 and 6 hpi, assessed by the two-sample Kolmogorov-Smirnov test.


( A ) mRNA expressions of PINK1 in CSCs non - CSCs . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O 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 expressions of PINK1 in CSCs and non-CSCs. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B - C ) Immunoblot of PINK1 in non - CSCs CSCs . Representative mean ± from 9 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O 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-C) Immunoblot analysis of PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 9 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D - E ) Immunoblot of p - PINK1 in non - CSCs CSCs . Representative mean ± from 7 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O 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-E) Immunoblot analysis of p-PINK1 protein in non-CSCs and CSCs. Representative and mean±SEM from 7 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Immunoblot of Parkin in non - CSCs CSCs .

Immunoblot analysis of Parkin protein in non-CSCs and CSCs.


of Parkin in non - CSCs CSCs . Representative mean ± from 8 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

analysis of Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H - I ) PINK1 Parkin in mitochondrial fractions whole lyses of CSCs non - CSCs . Representative mean ± from 8 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O 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-I) PINK1 and Parkin protein levels in mitochondrial fractions and whole cell lyses of CSCs and non-CSCs. Representative and mean±SEM from 8 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J - K ) Intracellular of p - Parkin in non - CSCs CSCs were analyzed using flow cytometry . Representative histograms mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(J-K) Intracellular levels of p-Parkin protein in non-CSCs and CSCs were analyzed using flow cytometry. Representative histograms and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


Immunoblot of p - Parkin in non - CSCs CSCs .

Immunoblot analysis of p-Parkin protein in non-CSCs and CSCs.


of p - Parkin in non - CSCs CSCs . Representative mean ± from 11 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

analysis of p-Parkin protein in non-CSCs and CSCs. Representative and mean±SEM from 11 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( N - P ) CSCs were treated with Mdivi - 1 ( 50 μM ) analyzed for sphere formation plus tumorgenicity . Mean ± from 6 experiments . Scale bar 20 mm . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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-EXP_ASSAY I-EXP_ASSAY 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

(N-P) CSCs were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Scale bar 20 mm. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( Q ) CSCs transfected with PINK1 shRNA or control were analysed for sphere formation tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(Q) CSCs transfected with PINK1 shRNA or the control were analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( R ) CSCs were treated with DAPT ( 10 μM ) analysed for sphere formation tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-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

(R) CSCs were treated with DAPT (10 μM) and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( S ) CSCs were transfected with PINK1 shRNA or control , analysed for activated Notch1 . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O 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 O O O O O O O O O O O O O O

(S) CSCs were transfected with PINK1 shRNA or control, and analysed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( T ) CSCs with or without transfection of Notch1 ICD were treated with Mdivi - 1 ( 50 μM ) analyzed for sphere formation plus tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(T) CSCs with or without transfection of Notch1 ICD were treated with Mdivi-1 (50 μM) and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A - B ) Lysosomal was identified with anti - LAMP1 anti - antibodies in non - CSCs CSCs . Scale bar 5μm . Representative mean ± with 50 from 5 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O 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-B) Lysosomal DNA was identified with anti-LAMP1 and anti-DNA antibodies in non-CSCs and CSCs. Scale bar 5μm. Representative and mean±SEM with 50 cells from 5 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) Lysosomal content of mtDNA in CSCs non - CSCs was determined with qPCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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

(C) Lysosomal content of mtDNA in CSCs and non-CSCs was determined with qPCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs were treated with Mdivi - 1 ( 50 μM ) analysed for lysosomal mtDNA . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-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 O O O O O O

(D) CSCs were treated with Mdivi-1 (50 μM) and analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) CSCs with or without transfection of PINK1 shRNA were analysed for lysosomal mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F - G ) expressions of TLR9 MyD88 in CSCs non - CSCs . Representative mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O 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-G) Protein expressions of TLR9 and MyD88 in CSCs and non-CSCs. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H ) TLR9 were immunoprecipitated from CSCs non - CSCs analyzed for binding load of mtDNA . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(H) TLR9 proteins were immunoprecipitated from CSCs and non-CSCs and analyzed for the binding load of mtDNA. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( I ) CSCs were transfected with mtDNA analysed for expressions of TLR9 plus MyD88 . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-SUBCELLULAR O O B-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 O O O O O O O O O O O O O

(I) CSCs were transfected with mtDNA and analysed for expressions of TLR9 plus MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J - K ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for lysosomal mtDNA , TLR9 , MyD88 . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR 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

(J-K) CSCs treated with EtBr (50 ng/ml) were analysed for lysosomal mtDNA, TLR9, and MyD88. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( L ) CSCs transfected with DNase II expression vector or control were analyzed for lysosomal plus MyD88 . Mean ± from 6 . ( M ) CSCs transfected with DNase II shRNA or control were analyzed for lysosomal plus MyD88 . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(L) CSCs transfected with DNase II expression vector or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. (M) CSCs transfected with DNase II shRNA or the control were analyzed for lysosomal DNA plus MyD88 protein. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) or control analysed for sphere formation tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-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

(A) CSCs were stimulated with CpG ODN (10 μg/ml) or control and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


CSCs transfected with mtDNA were analyzed for sphere formation Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-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

CSCs transfected with mtDNA were analyzed for sphere formation Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


CSCs transfected with mtDNA were analyzed for tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-SUBCELLULAR 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

CSCs transfected with mtDNA were analyzed for tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs were transfected with 2 different TLR9 shRNAs or control , analysed for sphere formation tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D) CSCs were transfected with 2 different TLR9 shRNAs or the control, and analysed for sphere formation and tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E - F ) CSCs transfected with DNase II expression vector , DNase II shRNA , or control was analyzed for sphere formation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E-F) CSCs transfected with DNase II expression vector, DNase II shRNA, or the control was analyzed for sphere formation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( G ) CpG promoted growth of PDX chimeras . Mean ± from 6 mice . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-SMALL_MOLECULE O B-EXP_ASSAY 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

(G) CpG promoted tumor growth of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( H ) TLR9 inhibitor chloroquine abrogated effect of CpG on growth in PDX chimeras . Mean ± from 6 mice . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(H) TLR9 inhibitor chloroquine abrogated the effect of CpG on tumor growth in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( I ) CpG treatment increased frequency of CD133 - expressing CSCs in PDX chimeras . Mean ± from 6 mice . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(I) CpG treatment increased the frequency of CD133-expressing CSCs in PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( J ) Chloroquine impeded function of CpG in elevating CD133 - expressing CSCs of PDX chimeras . Mean ± from 6 mice . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(J) Chloroquine impeded the function of CpG in elevating CD133-expressing CSCs of PDX chimeras. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs transfected with TLR9 shRNA or control were analysed for expressions of activated Notch1 plus HES1 . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) CSCs transfected with TLR9 shRNA or control were analysed for protein expressions of activated Notch1 plus HES1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B ) CSCs treated with or without EtBr ( 50 ng / ml ) were analyzed for activated Notch1 . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(B) CSCs treated with or without EtBr (50 ng/ml) were analyzed for activated Notch1. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) CSCs transfected with DNase II expression vector or control were analyzed for activated Notch1 expression. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs with or without CpG treatment were analysed for interaction between TLR9 Notch1 . Representative mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY 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 O O O O

(D) CSCs with or without CpG treatment were analysed for interaction between TLR9 and Notch1. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs were treated with CpG ODN ( 10 μg / ml ) in presence or absence of DAPT ( 10 μM ) , analyzed for sphere formation plus tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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

(E) CSCs were treated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F ) CSCs were transfected with 2 different Notch1 shRNAs or control , treated with CpG ODN ( 10 μg / ml ) , analyzed for sphere formation plus tumorgenicity . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(F) CSCs were transfected with 2 different Notch1 shRNAs or control, treated with CpG ODN (10 μg/ml), and analyzed for sphere formation plus tumorgenicity. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


PDX chimeras treated by CpG with or without DAPT were analysed for growth Mean ± from 6 mice . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY 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

PDX chimeras treated by CpG with or without DAPT were analysed for tumor growth Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


PDX chimeras treated by CpG with or without DAPT were analysed for CD133 - expressing CSCs . Mean ± from 6 mice . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

PDX chimeras treated by CpG with or without DAPT were analysed for CD133-expressing CSCs. Mean±SEM from 6 mice. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) CSCs non - CSCs were analyzed for mitochondrial metabolism using seahorse . Mean ± from 6 . Data information : Paired t - with Bonferroni method ( A , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-SUBCELLULAR 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

(A) CSCs and non-CSCs were analyzed for mitochondrial metabolism using seahorse assay. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method (A, **p < 0.01, ***p < 0.001.


( B ) CSCs were treated with 2 - DG ( 10 mM ) or Malonate ( 10 mM ) , assayed for sphere formation plus tumorgenicity . Mean ± from 6 . Data information : ANOVA with Turkey ' s method ( B ) . * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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

(B) CSCs were treated with 2-DG (10 mM) or Malonate (10 mM), and assayed for sphere formation plus tumorgenicity. Mean±SEM from 6 patients. Data information: ANOVA test with Turkey's method (B). **p < 0.01, ***p < 0.001.


( C ) CSCs treated with DAPT ( 10 μM ) were analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR 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

(C) CSCs treated with DAPT (10 μM) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( D ) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D) CSCs transfected with Notch1 shRNA or control shRNA were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( E ) CSCs treated with EtBr ( 50 ng / ml ) were analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR 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

(E) CSCs treated with EtBr (50 ng/ml) were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( F ) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

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

(F) CSCs transfected with DNase II expression vector or control were analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( G ) CSCs treated with chloroquine ( 50 μM ) were analyzed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method * * p < 0 . 01 , * * * p < 0 . 001 .

(G) CSCs treated with chloroquine (50 μM) were analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method **p < 0.01, ***p < 0.001.


( A ) CSCs stimulated with or without CpG ODN ( 10 μg / ml ) were analyzed for expression of phosphor - AMPKα . Representative mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-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

(A) CSCs stimulated with or without CpG ODN (10 μg/ml) were analyzed for expression of phosphor-AMPKα. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in presence or absence of DAPT ( 10 μM ) , analyzed for phosphor - AMPKα . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-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

(B) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of DAPT (10 μM), and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C ) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN ( 10 μg / ml ) analyzed for phosphor - AMPKα . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(C) CSCs transfected with Notch1 shRNA or control were stimulated with CpG ODN (10 μg/ml) and analyzed for phosphor-AMPKα. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( D ) CSCs were treated with DAPT ( 10 μM ) in presence or absence of A769662 ( 10 μM ) , analysed for mitochondrial OCR . Ctrl represents CSCs without any treatment . Mean ± from 6 . Data information : ANOVA with Turkey method ( D ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR 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

(D) CSCs were treated with DAPT (10 μM) in the presence or absence of A769662 (10 μM), and analysed for mitochondrial OCR. Ctrl represents CSCs without any treatment. Mean±SEM from 6 patients. Data information: ANOVA with Turkey method (D), **p < 0.01, ***p < 0.001, ****p < 0.0001.


( E ) CSCs were stimulated with or without CpG ODN ( 10 μg / ml ) analyzed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR 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

(E) CSCs were stimulated with or without CpG ODN (10 μg/ml) and analyzed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) CSCs were stimulated with CpG ODN ( 10 μg / ml ) in presence or absence of Compound C ( 10 μM ) analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR 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

(F) CSCs were stimulated with CpG ODN (10 μg/ml) in the presence or absence of Compound C (10 μM) and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( G ) CSCs were transfected with 2 different AMPKα shRNA or control , stimulated with CpG ODN ( 10 μg / ml ) , analysed for mitochondrial OCR . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(G) CSCs were transfected with 2 different AMPKα shRNA or the control, stimulated with CpG ODN (10 μg/ml), and analysed for mitochondrial OCR. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method , **p < 0.01, ***p < 0.001, ****p < 0.0001.


( A ) CSCs transfected with or without Notch1 ICD were analyzed for AMP / ATP plus LKB1 phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(A) CSCs transfected with or without Notch1 ICD were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( B ) CSCs treated with or without DAPT ( 10 μM ) were analyzed for AMP / ATP plus LKB1 phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(B) CSCs treated with or without DAPT (10 μM) were analyzed for AMP/ATP ratio plus LKB1 phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( C ) CSCs treated with or without CpG ODN ( 10 μg / ml ) were analysed for interactions between Notch1 AMPK . Representative mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY 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 O O O O

(C) CSCs treated with or without CpG ODN (10 μg/ml) were analysed for interactions between Notch1 and AMPK. Representative and mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( D ) CSCs transfected with LKB1 shRNA （ right ） or control ( left ) were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(D) CSCs transfected with LKB1 shRNA（right）or the control (left) were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( E ) CSCs transfected with TAK1 shRNA or control were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(E) CSCs transfected with TAK1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( F ) CSCs transfected with LAMTOR1 shRNA or control were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(F) CSCs transfected with LAMTOR1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( G ) CSCs transfected with AXIN1 shRNA or control were treated with CpG ODN ( 10 μg / ml ) analysed for AMPK phosphorylation . Mean ± from 6 . Data information : Paired t - with Bonferroni method , * * p < 0 . 01 , * * * p < 0 . 001 .

(G) CSCs transfected with AXIN1 shRNA or the control were treated with CpG ODN (10 μg/ml) and analysed for AMPK phosphorylation. Mean±SEM from 6 patients. Data information: Paired t-test with Bonferroni method, **p < 0.01, ***p < 0.001.


( A ) NSCLC PDX models treated with or without chemotherapy for first 2 were analyzed for growth at indicated time . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O B-DISEASE 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 O O O O 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) NSCLC PDX models treated with or without chemotherapy for the first 2 weeks were analyzed for tumor growth at the indicated time. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( B ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(B) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with or without chemotherapy were determined after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( C - E ) Flow cytometric analyses of expressions of TLR9 , activated Notch1 , phosphor - AMPKα in cancer from NSCLC xenografts treated with or without chemotherapy after 4 . Mean ± from 6 experiments . Data information : Paired t - with Bonferroni method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(C-E) Flow cytometric analyses of protein expressions of TLR9, activated Notch1, and phosphor-AMPKα in cancer cells from NSCLC xenografts treated with or without chemotherapy after 4 weeks. Mean±SEM from 6 experiments. Data information: Paired t-test with Bonferroni method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


( F ) NSCLC PDX models were treated with chemotherapy alone or together with indicated inhibitors , respectively , for first 2 . volume was determined at indicated time . Mean ± from 6 experiments . ( G ) Frequencies of CD133 - expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with indicated inhibitors after 4 . Mean ± from 6 experiments . Data information : ANOVA with Turkey ' s method . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(F) NSCLC PDX models were treated with chemotherapy alone or together with the indicated inhibitors, respectively, for the first 2 weeks. Tumor volume was determined at the indicated time. Mean±SEM from 6 experiments. (G) Frequencies of CD133-expressing CSCs from NSCLC xenografts treated with chemotherapy alone or with the indicated inhibitors after 4 weeks. Mean±SEM from 6 experiments. Data information: ANOVA test with Turkey's method. **p < 0.01, ***p < 0.001, ****p < 0.0001.


Cortical malformations observed in six novel BBSOAS ( P ) 1 shows abnormally elongated occipital convolutions ( arrowheads in A ) , whereas abnormal gyrification of supramarginal angular gyri is present in five out of six individuals ( P2 - P6 B ' , C ' C ' ' highlight abnormally convoluted regions boxed in B C .

B-TISSUE B-EXP_ASSAY O O O O B-DISEASE O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE 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

Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormally elongated occipital convolutions (arrowheads in A), whereas abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6 B', C' and C'' highlight the abnormally convoluted regions boxed in B and C.


Cortical malformations observed in six novel BBSOAS ( P ) 1 shows abnormal gyrification of supramarginal angular gyri is present in five out of six individuals ( P2 - P6 ;

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

Cortical malformations observed in six novel BBSOAS patients Patient (P)1 shows abnormal gyrification of supramarginal and angular gyri is present in five out of six individuals (P2-P6;


( H , H ' ) TBR2 ( green in H ) , SOX2 ( red in H ) NR2F1 ( yellow in H ' ) immunofluorescence ( IF ) of a GW11 section of human neocortex Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(H,H') TBR2 (green in H), SOX2 (red in H) and NR2F1 (yellow in H') immunofluorescence (IF) of a GW11 section of human neocortex Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.


( I ) NR2F1 expression ( quantified by pixel ) at different L - M in different regions along A - P extent of cortex , as indicated . n ≥ 3 sections from n = 1 brain . Data information number of positive was quantified in 100µm - width boxes , randomly placed across cortex . In graphs , data are represented as means ± . 2 - way ANOVA ( I ) ( * P < 0 . 05 , * * * P < 0 . 001 ) .

O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O 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 B-TISSUE O O O O O O 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 O O O O O O O O O O O O O O O

(I) NR2F1 expression (quantified by pixel intensity) at different L-M levels and in different regions along the A-P extent of the cortex, as indicated. n ≥ 3 sections from n = 1 fetal brain. Data information the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. 2-way ANOVA (I) (*P<0.05, ***P<0.001).


( J - L ' ) NR2F1 ( red ) IF of human GW14 neocortex , showing expression in CP ( K , L ) VZ ( K ' , L ' ) around convolutions in posterior - most cortex High NR2F1 is detected in neurogenic of a gyrus ( L ' ) . Data information : Nuclei ( blue ) were stained with DAPI . In Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; oSVZ : outer subventricular zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(J-L') NR2F1 (red) IF of human GW14 neocortex, showing expression levels in the CP (K,L) and VZ (K',L') around primary convolutions in the posterior-most cortex High NR2F1 is detected in the neurogenic area of a gyrus (L'). Data information: Nuclei (blue) were stained with DAPI. In Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; oSVZ: outer subventricular zone; SVZ: subventricular zone; VZ: ventricular zone.


( M , M ' ) NR2F1 level as measured on single in VZ ( M ) CP ( M ' ) of sulci gyri n ≥ 4 sections from n = 1 brain . ( N - O ) NR2F1 ( red ) TBR2 ( green ) immunostaining NR2F1 level as measured in single TBR2 + IPs by pixel is shown in graph ( O ) . n ≥ 4 convolutions from n = 1 brain ( n = 2 sections ) . Data information In graphs , data are represented as means ± . Student t - ( M , M ' , O ( * P < 0 . 05 , * * * P < 0 . 001 ) .

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

(M,M') NR2F1 level as measured on single cells in the VZ (M) and CP (M') of sulci and gyri n ≥ 4 sections from n = 1 fetal brain. (N-O) NR2F1 (red) and TBR2 (green) immunostaining NR2F1 level as measured in single TBR2+ IPs by pixel intensity is shown in graph (O). n ≥ 4 convolutions from n = 1 fetal brain (n = 2 sections). Data information In graphs, data are represented as means ± SEM. Student t-test (M,M',O (*P<0.05, ***P<0.001).


( P - R ) NR2F1 ( red ) HOPX ( green ) immunostaining Virtually HOPX + bRGs are also NR2F1 + ( magnification in P ' ' ) their number is greatly increased in gyri ( P ' ' ) compared to sulci ( P ' ) , as quantified in ( R ) . At a single level , NR2F1 pixel is higher in oSVZ HOPX + in gyri compared to sulci ( Q ) . n ≥ 4 sections from n = 1 brain . Data information : Nuclei ( blue ) were stained with DAPI . number of positive was quantified in 100µm - width boxes , randomly placed across cortex . In graphs , data are represented as means ± . Student t - Q , R ) ( * P < 0 . 05 , * * * P < 0 . 001 ) . oSVZ : outer subventricular

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

(P-R) NR2F1 (red) and HOPX (green) immunostaining Virtually all HOPX+ bRGs are also NR2F1+ (magnification in P'') and their number is greatly increased in gyri (P'') compared to sulci (P'), as quantified in (R). At a single cell level, NR2F1 pixel intensity is higher in oSVZ HOPX+ cells in gyri compared to sulci (Q). n ≥ 4 sections from n = 1 fetal brain. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the cortex. In graphs, data are represented as means ± SEM. Student t-test Q,R) (*P<0.05, ***P<0.001). oSVZ: outer subventricular zone


( A ) Representative of wild - type ( WT ) mutant ( KO ) neurospheres obtained from E15 . 5 neocortices cultured in vitro . culture passage ( " step " ) corresponds to 3 days of culture after dissociation re - plating . KO are more numerous larger in . Data information : Scale bars : 50µm .

O O O O O O O O O O O O O O O B-CELL_TYPE 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 O O O O O B-EXP_ASSAY O O O O O O O O O O O

(A) Representative images of wild-type (WT) and mutant (KO) neurospheres obtained from E15.5 neocortices and cultured in vitro. The culture passage ("step") corresponds to 3 days of culture after dissociation and re-plating. KO aggregates are more numerous and larger in size. Data information: Scale bars: 50µm.


( B ) Graph showing number of neurospheres per P100 plate at different steps , as indicated . Nr2f1KO neurospheres proliferate for a longer time ( up to 30 passages tested ) , whereas WT exhaust around step 9 . n ≥ 3 culture wells from n = 2 batches . Data information : data are represented as means ± . 2 - way ANOVA ( B ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(B) Graph showing the number of neurospheres per P100 cell plate at different steps, as indicated. Nr2f1KO neurospheres proliferate for a longer time (up to 30 passages tested), whereas WT cells exhaust around step 9. n ≥ 3 culture wells from n = 2 batches. Data information: data are represented as means ± SEM. 2-way ANOVA (B) (**P<0.01, ***P<0.001).


( C ) Panel of WT KO isolated neurospheres grown from day 1 to day 10 . Blue red lines represent diameter of WT KO neurospheres , respectively . Data information Scale bars : 50µm .

O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O

(C) Panel of WT and KO isolated neurospheres grown from day 1 to day 10. Blue and red lines represent the diameter of WT and KO neurospheres, respectively. Data information Scale bars: 50µm.


( D , E ) PH3 ( green ; dividing ) IF on cross - sections of day 4 neurospheres . arrowheads point at mitotic figures , which are quantified in ( E ) as of dividing over DAPI - stained nuclei ( blue ) . n ≥ 3 neurospheres from n = 2 batches . Data information : data are represented as means ± . Student t - ( E ) ( * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm .

O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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,E) PH3 (green; dividing cells) IF on cross-sections of day 4 neurospheres. White arrowheads point at mitotic figures, which are quantified in (E) as percentage of dividing cells over DAPI-stained nuclei (blue). n ≥ 3 neurospheres from n = 2 batches. Data information: data are represented as means ± SEM. Student t-test (E) (**P<0.01, ***P<0.001). Scale bars: 50µm.


( A - D ) Ki67 ( green ; mitotically active progenitors ) Tuj1 ( red ; post - mitotic neurons ) IF at E14 . 5 ( A , A ' ) , E16 . 5 ( B , B ' ) E18 . 5 ( C , C ' ) of lateral pallia of wild - type ( WT ) versus mutant ( KO ) brains . Ki67 + proliferating ( quantified in D ) accumulate in mutant cortices . n ≥ 3 brains . Data information : Nuclei ( blue ) were stained with DAPI . number of positive was quantified in 100µm - width boxes , randomly placed across LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(A-D) Ki67 (green; mitotically active progenitors) and Tuj1 (red; post-mitotic neurons) IF at E14.5 (A,A'), E16.5 (B,B') and E18.5 (C,C') of lateral pallia of wild-type (WT) versus mutant (KO) brains. Ki67+ proliferating cells (quantified in D) accumulate in mutant cortices. n ≥ 3 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


( E - F ' ) Tbr2 ( green ; IPs ) Pax6 ( red ; radial glia ) IF of E13 . 5 ( E , E ' ) E17 . 5 ( F , F ' ) lateral pallia of WT KO brains . Data information : Nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(E-F') Tbr2 (green; IPs) and Pax6 (red; radial glia cells) IF of E13.5 (E,E') and E17.5 (F,F') lateral pallia of WT and KO brains. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


of different classes of cortical progenitors : apical radial glia ( aRGs ) as Pax6 + NPs in VZ ( G ) , basal intermediate progenitors ( IPs ) as Tbr2 + in SVZ ( H ) n ≥ 3 brains . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

Quantification of different classes of cortical progenitors: apical radial glia cells (aRGs) as Pax6+ NPs in VZ (G), basal intermediate progenitors (IPs) as Tbr2+ cells in SVZ (H) n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


of different classes of cortical progenitors : , basal RGs as Pax6 + in outer SVZ ( I ) . n ≥ 3 brains . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across LP . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

Quantification of different classes of cortical progenitors: , basal RGs as Pax6+ cells in outer SVZ (I). n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( J - L ) E12 . 5 dorsal brain views ( J , J ' ) representative sections of posterior hemispheres ( K , K ' ) , showing a vesicle enlargement in mutant brains ( K ' ) . extension of ventricular surface was quantified along A - P axis ( L ) ; red color code represents Nr2f1 gradient in WT brains . 12µm - thick sections were collected on series of 10 slides ; consecutive measurement are 120µm apart from each other . n ≥ 3 brains . Data information : In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(J-L) E12.5 dorsal brain views (J,J') and representative sections of posterior hemispheres (K,K'), showing a vesicle enlargement in mutant brains (K'). The extension of the ventricular surface was quantified along the A-P axis (L); red color code represents Nr2f1 gradient in WT brains. 12µm-thick sections were collected on series of 10 slides; consecutive measurement levels are 120µm apart from each other. n ≥ 3 brains. Data information: In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( N - P ) GFP ( green ; expressed under Sox2 promoter ) RFP ( red ; expressed under Tis21 promoter ) IF of E13 . 5 lateral pallia , 18 - hours after IUE . proportion of single or double positive NPs is shown in pie charts in ( P ) . n = 3 electroporated brains . Data information : Nuclei ( blue ) were stained with DAPI . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(N-P) GFP (green; expressed under Sox2 promoter) and RFP (red; expressed under Tis21 promoter) IF of E13.5 lateral pallia, 18-hours after IUE. The proportion of single or double positive NPs is shown in pie charts in (P). n = 3 electroporated brains. Data information: Nuclei (blue) were stained with DAPI. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( Q - R ' ) Ki67 ( green ; progenitors ) EdU ( red ) IF of E13 . 5 WT ( Q , Q ' ) KO ( R , R ' ) , injected with EdU at E12 . 5 . Differentiating ( EdU + Ki67 - ) are located in IZ / CP ; percentages shown in ( Q ' , R ' ) were obtained from n ≥ 4 sections of n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . Student t - ( Q ' , R ' ; E13 . 5 , EdU injected at E12 . 5 ; WT / KO : * = 0 . 03967 ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . Scale bars : 50µm . CP : cortical plate ; IZ : intermediate zone ; SVZ : subventricular zone ; VZ : ventricular zone .

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

(Q-R') Ki67 (green; progenitors) and EdU (red) IF of E13.5 WT (Q,Q') and KO (R,R') embryos, injected with EdU at E12.5. Differentiating cells (EdU+Ki67-) are located in the IZ/CP; percentages shown in (Q',R') were obtained from n ≥ 4 sections of n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. Student t-test (Q',R'; E13.5, EdU injected at E12.5; WT/KO: *=0.03967 (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.


( A - D ) Graphs showing average number of PH3 + mitotic figures in posterior lateral pallium of WT mutant cortices . total number of mitoses ( A ) as well as number of dividing in VZ ( Pax6 + Sox2 + aRGs ; B ) , SVZ ( Tbr2 + IPs ; C ) outer ( basal - most ) region of SVZ ( oSVZ / IZ ; Pax6 + oRGs ; D ) are shown . For graphs ( B - D ) , statistical by 2 - way ANOVA n ≥ 3 brains per / genotype . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . SVZ : subventricular zone ; VZ : ventricular zone .

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

(A-D) Graphs showing the average number of PH3+ mitotic figures in the posterior lateral pallium of WT and mutant cortices. The total number of mitoses (A) as well as the number of dividing cells in VZ (Pax6+Sox2+ aRGs; B), SVZ (Tbr2+ IPs; C) and outer (basal-most) region of SVZ (oSVZ/IZ; Pax6+ oRGs; D) are shown. For graphs (B-D), statistical analysis by 2-way ANOVA n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). SVZ: subventricular zone; VZ: ventricular zone.


( E ) cycle duration at different embryonic ages ( from E10 . 5 to E16 . 5 ) in WT ( blue bars ) or KO ( orange ) , as quantified upon a double EdU / BrdU injection protocol in posterior - most LP n ≥ 3 brains per / genotype . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O B-EXP_ASSAY 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 O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O 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) Cell cycle duration at different embryonic ages (from E10.5 to E16.5) in WT (blue bars) or KO embryos (orange line), as quantified upon a double EdU/BrdU injection protocol in posterior-most LP n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).


( F - G ' ) Ki67 ( green ; NPs ) EdU ( red ) IF in WT ( F , F ' ) KO ( G , G ' ) after 6 hours consecutive EdU incorporation . Note higher number of labelled NPs in KO cortices ( G ' ) suggesting faster cycle progression / or longer S - phase . Data information : Nuclei ( blue ) were stained with DAPI .

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

(F-G') Ki67 (green; NPs) and EdU (red) IF in WT (F,F') and KO (G,G') after 6 hours consecutive EdU incorporation. Note the higher number of labelled NPs in KO cortices (G') suggesting faster cell cycle progression and/or longer S-phase. Data information: Nuclei (blue) were stained with DAPI.


( H ) Best linear fit of EdU + Ki67 + NP after 8 hours EdU cumulative labelling in WT ( blue ) KO ( orange ) brains . x - y - intercept are proportional to S - phase to number of cycling , respectively , while steepness of is proportional to cycle duration . WT : y = 11 . 66x + 24 . 62 ; KO : y = 13 . 54x + 32 . 96 ) . n ≥ 2 brains per time point / genotype . Data information : number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . 2 - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O 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) Best linear fit of EdU+Ki67+ NP percentage after 8 hours EdU cumulative labelling in WT (blue line) and KO (orange line) brains. The x- and y-intercept are proportional to the S-phase length and to the number of cycling cells, respectively, while the steepness of the line is proportional to cell cycle duration. WT: y = 11.66x +24.62; KO: y = 13.54x +32.96). n ≥ 2 brains per time point/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001).


( I - K ) PH3 ( green ; dividing NPs ) Pax6 ( red ; aRGs ) IF of E13 . 5 WT ( I ) KO ( J ) lateral pallia . Strong weak / punctate PH3 patterns show M - phase G2 - phase , respectively . G2 - phase NP is quantified in ( K ) . As a result of shorter G1 - phase globally shorter cycle time , of G2 - phase is increased in KO NP pool . n ≥ 2 brains . Data information : Nuclei ( blue ) were stained with DAPI . number of positive was quantified in 100µm - width boxes , randomly placed across lateral pallium . In graphs , data are represented as means ± . Student t - ( K ; * P < 0 . 05 ) Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone .

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

(I-K) PH3 (green; dividing NPs) and Pax6 (red; aRGs) IF of E13.5 WT (I) and KO (J) lateral pallia. Strong and weak/punctate PH3 patterns show M-phase and G2-phase cells, respectively. G2-phase NP percentage is quantified in (K). As a result of shorter G1-phase and globally shorter cell cycle time, the percentage of G2-phase cells is increased in the KO NP pool. n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. Student t-test (K; *P<0.05) Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.


( L ) cycle time of E13 . 5 NPs in lateral pallia of WT ( upper column ) KO ( lower column ) , as calculated by EdU cumulative labelling PH3 KO NPs have a 16 . 1 % faster cycle time , due to a 30 . 9 % shorter G1 - phase compensating a 14 . 3 % longer S - phase . n ≥ 3 brains . In graphs , data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(L) Cell cycle time of E13.5 NPs in the lateral pallia of WT (upper column) and KO cells (lower column), as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16.1% faster cell cycle time, due to a 30.9% shorter G1-phase compensating a 14.3% longer S-phase. n ≥ 3 brains. In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).


( A - B ' ) Pax6 ( green ; aRGs ) Nr2f1 ( red ) IF of E12 . 5 WT ( A , A ' ) KO ( B , B ' ) lateral pallia . Data information : nuclei ( blue ) were stained with DAPI . Scale bars : 50µm . SVZ : subventricular zone ; VZ : ventricular zone .

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

(A-B') Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A,A') and KO (B,B') lateral pallia. Data information: nuclei (blue) were stained with DAPI. Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.


( C ) Pax6 Nr2f1 pixel at E12 . 5 E14 . 5 indicating increased Pax6 upon Nr2f1 removal . n ≥ 3 brains . Data information : pixel was quantified in 100µm - width boxes , randomly placed across lateral pallium . Data are represented as means ± . 2 - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O 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) Pax6 and Nr2f1 pixel intensity quantification at E12.5 and E14.5 indicating increased Pax6 levels upon Nr2f1 removal. n ≥ 3 brains. Data information: the pixel intensity was quantified in 100µm-width boxes, randomly placed across the lateral pallium. Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).