a , Time - lapse of RPE1 WT SEPT2 - KO stably expressing ARL13B - GFP γ - tubulin - mRuby2 . are starved for 32h before beginning of inspection imaged every 15min for 9 . 5h . Scale bar : 5µm . b , of ( a ) showing changes in cilia during time - lapse imaging . Three biological replicates , 10 cilia per sample repetition . c , Time - lapse show ectocytosis events observed in RPE1 SEPT2 - KO . Experiments are done as in ( a ) with exception that are imaged every 15min . Scale bar : 2 . 5µm .

O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O 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, Time-lapse images of RPE1 WT and SEPT2-KO cells stably expressing ARL13B-GFP and γ-tubulin-mRuby2. Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. Scale bar: 5µm. b, Quantification of (a) showing changes in cilia length during time-lapse imaging. Three biological replicates, 10 cilia per sample and repetition. c, Time-lapse images show ectocytosis events observed in RPE1 SEPT2-KO cells. Experiments are done as in (a) with exception that cells are imaged every 15min. Scale bar: 2.5µm.


g - h , RPE1 SEPT2 - KO expressing ARL13B - mRuby2 Neongreen - EFHC1 after 48h SS immunostained with Glu - tub antibody . Representative ( g ) quantifications of proximal segment ( Glu tub ) , EFHC1 whole cilia ( ARL13B ) ( h ) are shown . Three biological replicates , n = 100 cilia per sample repetition . Scale bar : 2µm .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR 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-h, RPE1 SEPT2-KO cells expressing ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained with Glu-tub antibody. Representative images (g) and quantifications of proximal segment (Glu tub), EFHC1 and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm.


mice , housed at 30 ° C or 6 ° C for 10 days , were intraperitoneally ( IP ) administered with [ U - 13C ] glucose ( 2 g / kg ) . 15 minutes after injection , BAT was harvested for metabolic enrichment . ( A ) m + 6 glucose enrichment in serum BAT . ( B ) relative glucose abundance in serum BAT .

O O O 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-MEASURED_VAR O 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-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O

Male mice, housed at 30°C or 6°C for 10 days, were intraperitoneally (IP) administered with [U-13C]glucose (2 g/kg). 15 minutes after injection, BAT was harvested for metabolic enrichment assay. (A) m+6 glucose enrichment in serum and BAT. (B) relative glucose abundance in serum and BAT.


( D , E ) Metabolic 13C enrichments in BAT of mice are shown as m + 3 glycolytic intermediates ( D ) , m + 2 TCA cycle intermediates ( E ) .

O O O O O O O O O O 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) Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates (D), m+2 TCA cycle intermediates (E).


( A ) Representative immunofluorescence shows UCP1 ( green ) , perilipin ( red ) DAPI ( blue ) in sWAT of mice housed at 6 ° C .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(A) Representative immunofluorescence staining shows UCP1 (green), perilipin (red) and DAPI (blue) in sWAT of mice housed at 6°C.


( B - G ) mice , housed at 30 ° C or 6 ° C for 10 days , were administered with [ U - 13C ] glucose ( 2 g / kg , IP ) . 15 minutes after injection , sWAT gWAT were harvested for metabolic enrichment . Metabolic 13C enrichments in sWAT of mice are shown as m + 6 glucose ( B ) , m + 3 glycolytic intermediates ( C ) , m + 2 TCA cycle intermediates ( D ) . Metabolic 13C enrichments in gWAT of mice are shown as m + 6 glucose ( E ) , m + 3 glycolysis intermediates ( F ) , m + 2 TCA cycle intermediates ( G ) .

O O O O 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-MEASURED_VAR O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(B-G) Male mice, housed at 30°C or 6°C for 10 days, were administered with [U-13C]glucose (2 g/kg, IP). 15 minutes after injection, sWAT and gWAT were harvested for metabolic enrichment assay. Metabolic 13C enrichments in sWAT of male mice are shown as m+6 glucose (B), m+3 glycolytic intermediates (C), and m+2 TCA cycle intermediates (D). Metabolic 13C enrichments in gWAT of male mice are shown as m+6 glucose (E), m+3 glycolysis intermediates (F), and m+2 TCA cycle intermediates (G).


( H ) Plasma 13CO2 enrichment , after normalizing to [ U - 13C ] glucose tracer enrichment .

(H) Plasma 13CO2 enrichment, after normalizing to the [U-13C]glucose tracer enrichment.


Mouse brown adipocytes were differentiated from freshly isolated in stromal vascular fractions ( SVF ) of interscapular BAT . ( A ) Upper : Oil red of during differentiation . Lower : Relative mRNA of brown adipocyte markers were measured by qPCR .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (A) Upper: Oil red staining of the cells during differentiation. Lower: Relative mRNA levels of brown adipocyte markers were measured by qPCR.


Mouse brown adipocytes were differentiated from freshly isolated in stromal vascular fractions ( SVF ) of interscapular BAT . ( B ) Glycolytic intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (B) Glycolytic intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


Mouse brown adipocytes were differentiated from freshly isolated in stromal vascular fractions ( SVF ) of interscapular BAT . ( C ) TCA cycle intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (C) TCA cycle intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


( A ) Fully differentiated brown adipocytes were treated with β3 - AR agonist CL316 , 243 ( 10 µM ) . Relative mRNA of thermogenic markers were measured by qPCR .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

(A) Fully differentiated brown adipocytes were treated with β3-AR agonist CL316,243 (10 µM). Relative mRNA levels of thermogenic markers were measured by qPCR.


( B ) Pyruvate lactate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose 10 µM CL316 , 243 for 2 hours .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

(B) Pyruvate and lactate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( C ) Citrate malate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose 10 µM CL316 , 243 for 2 hours .

(C) Citrate and malate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( A ) oxygen consumption rate ( OCR ) of mouse brown adipocytes treated with MPC inhibitor CHC ( 2 mM ) or UK5099 ( 2 µM ) , n = 6 - 7 biological repeats .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats.


( B ) Differentiated mouse brown adipocytes were pre - treated with β3 - AR agonist CL316 , 243 for 4 hours , before cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours with CHC or UK5099 . Metabolic 13C enrichments in brown adipocytes were shown as m + 3 glycolytic intermediates , m + 2 m + 3 TCA cycle intermediates , n = 3 biological replicates .

O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O 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) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates.


( C ) After housed at 6 ° C for 10 days , mice were IP injected with PBS or CHC ( 500 mg / kg ) . 30 minutes after CHC treatment , mice were IP administered with [ U - 13C ] glucose ( 2 g / kg ) . Metabolic 13C enrichments in BAT of mice are shown as m + 3 glycolytic intermediates , m + 2 m + 3 TCA cycle intermediates , n = 7 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7.


( D ) of CHC ( left : 400 mg / kg , n = 5 ) ( right : 500 mg / kg , n = 7 ) treated mice .

O O O O B-CONTROLLED_VAR O O O 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) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.


B Elution volumes ( peak positions ) of Munc18 - 1 mutants in absence ( top ) presence ( bottom ) of Syx1 using - exclusion chromatography on Superdex - 200 column .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

B Elution volumes (peak positions) of the Munc18-1 mutants in the absence (top) and presence (bottom) of Syx1 using size-exclusion chromatography on Superdex-200 column.


C Effects of domain 3a mutations on MUN - catalyzed transition detected by native - PAGE . Components addition was indicated on top of every lane . D Quantitative of integrated densities of Munc18 - 1 / Syx1 bands in C .

O O O O O O O B-CONTROLLED_VAR O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O

C Effects of the domain 3a mutations on MUN-catalyzed transition detected by native-PAGE assay. Components addition was indicated on the top of every lane. D Quantitative analysis of the integrated densities of Munc18-1/Syx1 bands in C.


Effects of QKAA , KKEE L348R mutations on MUN - catalyzed transition detected by FRET . Schematic diagram is shown on top of chart . FRET between BODIPY FL - labeled Syb2 ( donor ) TMR - labeled SN25 ( acceptor ) was monitored .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O

Effects of the QKAA, KKEE and L348R mutations on MUN-catalyzed transition detected by FRET assay. Schematic diagram is shown on the top of the chart. FRET between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.


F Effects of QKAA , KKEE L348R mutations on lipid mixing between liposomes with Munc18 - 1 / Syx1 liposomes containing Syb2 in presence of Munc13 ( M13 ) , SN25 Syt1 / Ca2 + . Illustration of reactions is shown on top of chart .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR B-MEASURED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

F Effects of the QKAA, KKEE and L348R mutations on lipid mixing between liposomes with Munc18-1/Syx1 and liposomes containing Syb2 in the presence of Munc13 (M13), SN25 and Syt1/Ca2+. Illustration of the reactions is shown on the top of the chart.


A Representative traces ( left ) , summary graphs of normalized frequency ( middle ) normalized amplitude ( right ) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18 - 1 WT or mutants , with or without Munc18 - 1 shRNAs ( Control , n = 25 ; None , n = 17 ; WT , n = 17 ; QKAA , n = 18 ; KKEE , n = 16 ; P335A , n = 23 ; L348R , n = 21 ) . B Sample traces ( left ) , summary graphs of normalized amplitude ( middle ) normalized charge transfer ( right ) of evoked IPSCs recorded from neurons as described in panel A ( Control , n = 19 ; None , n = 19 ; WT , n = 19 ; QKAA , n = 13 ; KKEE , n = 18 ; P335A , n = 17 ; L348R , n = 21 ) . C Sample traces ( left ) of normalized charge transfer evoked by hypertonic sucrose ( right ) recorded from neurons described in panel A ( Control , n = 17 ; None , n = 13 ; WT , n = 17 ; QKAA , n = 17 ; KKEE , n = 18 ; P335A , n = 15 ; L348R , n = 14 ) .

O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21). B Sample traces (left), summary graphs of normalized amplitude (middle) and normalized charge transfer (right) of evoked IPSCs recorded from neurons as described in panel A (Control, n = 19; None, n = 19; WT, n = 19; QKAA, n = 13; KKEE, n = 18; P335A, n = 17; L348R, n = 21). C Sample traces (left) and quantification of the normalized charge transfer evoked by hypertonic sucrose (right) recorded from the neurons described in panel A (Control, n = 17; None, n = 13; WT, n = 17; QKAA, n = 17; KKEE, n = 18; P335A, n = 15; L348R, n = 14).


A Representative traces ( left ) , summary graphs of normalized frequency ( middle ) normalized amplitude ( right ) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18 - 1 WT or mutants , with or without Munc18 - 1 shRNAs ( Control , n = 25 ; None , n = 17 ; WT , n = 17 ; QKAA , n = 18 ; KKEE , n = 16 ; P335A , n = 23 ; L348R , n = 21 ) .

O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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 Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21).


A Representative of HEK293T transfected with mCherry2 - Syx1 or EGFP - Munc18 - 1 . Lipophilic dye DiD ( 1 , 1 - Dioctadecyl - 3 , 3 , 3 , 3 - tetramethylindodicarbocyanine ) was used as plasma marker . B Representative of HEK293T co - transfected with mCherry2 - Syx1 EGFP labeled WT or mutants of Munc18 - 1 .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

A Representative images of HEK293T cells transfected with mCherry2-Syx1 or EGFP-Munc18-1. Lipophilic dye DiD (1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) was used as the plasma membrane marker. B Representative images of HEK293T cells co-transfected with mCherry2-Syx1 and EGFP labeled WT or mutants of Munc18-1.


C , D Summary graphs of distribution of Syx1 ( C ) Munc18 - 1 WT or mutants ( D ) quantified by of at plasma ( PM ) to total in whole ( total ) . DiD is PM marker . Numbers of pictures analyzed are shown as follows : no Syx1 , n = 14 ; no M18 , n = 15 ; WT , n = 24 ; QKAA , n = 23 ; KKEE , n = 12 ; L348R , n = 22 .

O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O 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,D Summary graphs of the membrane distribution of Syx1 (C) and Munc18-1 WT or mutants (D) quantified by the ratio of fluorescence at the plasma membrane (PM) to the total fluorescence in the whole cell (total). DiD is the PM marker. Numbers of pictures analyzed are shown as follows: no Syx1, n = 14; no M18, n = 15; WT, n = 24; QKAA, n = 23; KKEE, n = 12; L348R, n = 22.


E Munc18 - 1 mutants retain interaction with Syx1 shown by GST pull - down experiment . A representative gel displayed is from one of three independent replicates .

O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.


B Example histograms of smFRET efficiency of Syx1 in with Munc18 - 1 WT in absence presence of MUN domain , within SNARE . C − E Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 QKAA ( C ) , KKEE ( D ) , L348R ( E ) in absence or presence of MUN domain . QKAA mutation strongly impaired shift of smFRET efficiency in presence of MUN domain .

O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O

B Example histograms of smFRET efficiency of Syx1 in complex with Munc18-1 WT in the absence and presence of the MUN domain, and within the SNARE complex. C−E Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 QKAA (C), KKEE (D), and L348R (E) in the absence or presence of the MUN domain. The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain.


F Quantitative results of Gaussian peak center positions of FRET efficiency distributions in C − E .

F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C−E.


G Effects of QKAA , KKEE , L348R mutations on interaction between Munc18 - 1 / Syx1 MUN domain by GST pull - down combined with immunoblotting . Results were shown with 6 × His - tag antibody GST - tag antibody .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O 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 Effects of QKAA, KKEE, and L348R mutations on the interaction between the Munc18-1/Syx1 complex and the MUN domain by GST pull-down assay combined with immunoblotting. Results were shown with 6×His-tag antibody and GST-tag antibody.


B Effects of QKAA , KKEE , L348R mutations on binding of Syb2 to Munc18 - 1 / Syx1 in presence absence of MUN domain detected by GST pull - down . Bands of Munc18 - 1 are indicated with red asterisks . C of normalized binding of Syb2 to Munc18 - 1 / Syx1 . Integrated intensities of Munc18 - 1 bands were quantified . QKAA L348R , but not KKEE , disrupted Syb2 binding to Munc18 - 1 / Syx1 .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O

B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks. C Quantification of the normalized binding of Syb2 to Munc18-1/Syx1 complex. Integrated intensities of Munc18-1 bands were quantified. QKAA and L348R, but not KKEE, disrupted Syb2 binding to the Munc18-1/Syx1 complex.


B Effects of QKAA , KKEE , L348R mutations on binding of Syb2 to Munc18 - 1 / Syx1 in presence absence of MUN domain detected by GST pull - down . Bands of Munc18 - 1 are indicated with red asterisks .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks.


A Ensemble FRET showing different effects of Munc18 - 1 P335A mutation Syx1 LEAA mutation in transition from Munc18 - 1 / Syx1 to SNARE with without MUN domain . FRET signal between BODIPY FL - labeled Syb2 ( donor ) TMR - labeled SN25 ( acceptor ) was monitored . B of FRET results in A .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored. B Quantification of the FRET results in A.


A Ensemble FRET showing different effects of Munc18 - 1 P335A mutation Syx1 LEAA mutation in transition from Munc18 - 1 / Syx1 to SNARE with without MUN domain . FRET signal between BODIPY FL - labeled Syb2 ( donor ) TMR - labeled SN25 ( acceptor ) was monitored .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O

A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.


C Single - molecule FRET showing MUN domain drives open of Syx1 linker region independent of conformational change of domain 3a . Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 P335A mutant in absence presence of MUN domain . D Quantitative results of Gaussian peak center positions of FRET efficiency distributions in C .

O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain. D Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C.


C Single - molecule FRET showing MUN domain drives open of Syx1 linker region independent of conformational change of domain 3a . Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 P335A mutant in absence presence of MUN domain .

O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O

C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain.


B Munc18 - 1 , Habc H3 construct a tertiary .

O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

B Munc18-1, Habc and H3 construct a tertiary complex.


C tertiary globally resembles Munc18 - 1 / Syx1 .

C The tertiary complex globally resembles the Munc18-1/Syx1 complex.


D Formation of tertiary with Munc18 - 1 QKAA , KKEE , L348R mutants . KKEE strongly impaired formation of tertiary .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

D Formation of the tertiary complex with Munc18-1 QKAA, KKEE, and L348R mutants. KKEE strongly impaired formation of the tertiary complex.


E Munc18 - 1 displays interaction with Syx1 - Habc .

E Munc18-1 displays interaction with Syx1-Habc.


F QKAA , KKEE , L348R retained interaction with Syx1 - Habc .

F QKAA, KKEE, and L348R all retained interaction with Syx1-Habc.


( B ) NSC - 34 SH - SY5Y were transfected with various concentrations of siPRPH or siNTC . efficiency of siRNA knockdown at 48h . p . t . was validated by Western blot while cytotoxicity was determined by AlamarBlue . siPRPH - or siNTC - transfected were infected with EV - A71 S41 , viral titers were determined at 48h . p . i .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NSC-34 and SH-SY5Y cells were transfected with various concentrations of siPRPH or siNTC. The efficiency of siRNA knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay. siPRPH- or siNTC-transfected cells were infected with EV-A71 S41, and the viral titers were determined at 48h.p.i.


Two - week old AG129 mice ( n = 2 - 3 / group / time point ) were infected with EV - A71 S41 . ( A ) At various time points post - infection , mice were euthanized relative expression of PRPH was detected by Western blot in tissue homogenates ( brainstem , spinal cord limb muscle ) . Actin GAPDH serve as loading controls , expression was compared to - matched non - infected mice .

O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (A) At various time points post-infection, mice were euthanized and the relative expression of PRPH was detected by Western blot in tissue homogenates (brainstem, spinal cord and limb muscle). Actin and GAPDH serve as loading controls, and protein expression was compared to age-matched non-infected mice.


Two - week old AG129 mice ( n = 2 - 3 / group / time point ) were infected with EV - A71 S41 . ( B ) Immunostaining of PRPH EV - A71 particles on brainstem spinal cord sections harvested at 6 d . p . i .

O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O

Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (B) Immunostaining of PRPH and EV-A71 particles on brainstem and spinal cord sections harvested at 6 d.p.i.


( C ) Proximity ligation . NSC - 34 SH - SY5Y were infected with EV - A71 S41 at MOI 10 0 . 01 , respectively . were fixed at 48h . p . i . prior to PLA using anti - PRPH anti - VP0 antibodies .

O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O B-MEASURED_VAR O O

(C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. Cells were fixed at 48h.p.i. prior to PLA staining using anti-PRPH and anti-VP0 antibodies.


( A ) NSC - 34 SH - SY5Y were reverse - transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV - A71 . culture supernatants were harvested for viral titer determination at 24h . p . t .

O O O O O O O O O O O O O O O O O O O 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) NSC-34 and SH-SY5Y cells were reverse-transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV-A71 RNA. The culture supernatants were harvested for viral titer determination at 24h.p.t.


( A ) NSC - 34 SH - SY5Y were infected with EV - A71 S41 at MOI 30 0 . 01 , respectively . At 48 h . p . i . , were fixed permeabilized prior to immunostaining .

O O O O O O O O O O O O O O O O O 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) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48 h.p.i., cells were fixed and permeabilized prior to immunostaining.


( B ) Total infected lysates were pulled down with anti - PRPH or IgG isotype control antibody prior to immunoblotting . Uninfected were included as negative control . IP : immunoprecipitation ; IB : immunoblotting .

O O O O O O O O O O O O B-MEASURED_VAR 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) Total infected cell lysates were pulled down with anti-PRPH or IgG isotype control antibody prior to immunoblotting. Uninfected cells were included as negative control. IP: immunoprecipitation; IB: immunoblotting.


C ) Proximity ligation . NSC - 34 SH - SY5Y were infected with EV - A71 S41 at MOI 10 0 . 01 , respectively . At 48 h . p . i . , were fixed permeabilized prior to PLA using antibodies specific to PPRH , VP0 , 3C 3D .

O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O

C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. At 48 h.p.i., the cells were fixed and permeabilized prior to PLA staining using antibodies specific to PPRH, VP0, 3C and 3D.


( C ) Viral titers in siRNA - treated NSC - 34 . were transfected with various concentrations of siRNA specific to indicated target genes . efficiency of siRNA - mediated knockdown at 48h . p . t . was validated by Western blot while cytotoxicity was determined by AlamarBlue siRNA - or siNTC - transfected were infected with EV - A71 S41 at MOI 10 , viral titers were determined at 48h . p . i .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Viral titers in siRNA-treated NSC-34 cells. Cells were transfected with various concentrations of siRNA specific to the indicated target genes. The efficiency of siRNA-mediated knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay siRNA- or siNTC-transfected cells were infected with EV-A71 S41 at MOI 10, and the viral titers were determined at 48h.p.i.


( D ) Drug treatment . EV - A71 - infected NSC - 34 were treated with indicated drugs , viral titers were determined at 48h . p . i .

O O O O O O O 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) Drug treatment. EV-A71-infected NSC-34 cells were treated with the indicated drugs, and the viral titers were determined at 48h.p.i.


( A ) NSC - 34 SH - SY5Y were infected with EV - A71 S41 at MOI 30 0 . 01 , respectively . At 48h . p . i . , were fixed permeabilized prior to immunostaining .

O O O O O O O O O O O O O O O O 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) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48h.p.i., the cells were fixed and permeabilized prior to immunostaining.


B ) Proximity ligation . NSC - 34 SH - SY5Y were transfected with LucEV - A71 replicon . were fixed permeabilized at 24h . p . t . prior to PLA using antibodies specific to Rac1 , 3C 3D .

O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O

B) Proximity ligation assay. NSC-34 and SH-SY5Y cells were transfected with LucEV-A71 replicon. Cells were fixed and permeabilized at 24h.p.t. prior to PLA staining using antibodies specific to Rac1, 3C and 3D.


of total - free ( cfDNA ) in 13 CSF samples . Samples in which SCNAs were detected are shown in blue , tended to have higher of total

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Concentration of total cell-free DNA (cfDNA) in 13 CSF samples. Samples in which SCNAs were detected are shown in dark blue, and tended to have higher levels of total cfDNA


Relative copy number estimation in 20 genes of interest , 18 large genomic regions , determined by sWGS of CSF from 13 glioma . Genes are ordered by genomic position , their chromosomal locations are indicated in parentheses . Amplifications are shown in blue , deletions in orange , copy number neutral regions are in grey . top green bar indicates largest dimension ( < 42 mm vs > 42 mm ) , top violet bar indicates glioma subtype . SCNAs were more frequently detected in CSF from with large glioblastoma

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

Relative copy number estimation in 20 genes of interest, and 18 large genomic regions, determined by sWGS of CSF from 13 glioma patients. Genes are ordered by genomic position, and their chromosomal locations are indicated in parentheses. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in grey. The top green bar indicates the tumor largest length dimension (<42 mm vs >42 mm), and the top violet bar indicates the glioma subtype. SCNAs were more frequently detected in CSF from patients with large glioblastoma tumors


SCNAs determined by sWGS in 4 subparts ( T1 - T4 ) CSF sample , collected from G1 . Amplifications are shown in blue , deletions in orange , copy number neutral regions are in grey . sWGS from plasma urine samples collected at same time as CSF sample showed no

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

SCNAs determined by sWGS in 4 tumor subparts (T1 - T4) and the CSF sample, collected from patient G1. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in dark grey. sWGS from plasma and urine samples collected at the same time as the CSF sample showed no SCNAs


Heat map summarizing SCNAs detected by sWGS of 28 genes of interest in biopsies CSF from G1 ( 4 subparts 1 CSF sample ) . Amplifications are shown in blue , deletions in orange , copy number neutral regions are in

Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 (4 tumor subparts and 1 CSF sample). Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in light grey


Heat map summarizing detection of EGFR PTEN alterations in tissue in CSF samples . Shared detection in tissue CSF is indicated in green , detection of alteration only in tissue in orange , non - detection in blue . top bars indicate cfDNA ( copies / mL ; in a range of purples ) , of tumors ( in a range of browns ) , type of glioma ( in a range of blues ) , whether was in direct contact with CSF or not ( on MRI , green or red , respectively ) . Samples are ranked from left to right by decreasing of cfDNA ( copies / mL )

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples. Shared detection in tissue and CSF is indicated in green, detection of the alteration only in tissue in orange, and non-detection in blue. The top bars indicate the cfDNA concentration (copies/mL; in a range of purples), the size of the tumors (in a range of browns), the type of glioma (in a range of blues), and whether the tumor was in direct contact with the CSF or not (based on MRI, green or red, respectively). Samples are ranked from the left to right by decreasing concentration of cfDNA (copies/mL)


A ) Fragment distribution , determined by sWGS , in CSF ( blue ) , plasma ( red dashed ) urine ( black dashed ) from G1 . 3 samples were collected simultaneously before initiation of treatment . B ) Fragment distribution of cfDNA , determined by sWGS , in CSF from 13 glioma . samples contained a high fraction of reads from fragments with lengths corresponding to ~ 145 bp ~ 167 bp . fragment profile of cfDNA from plasma sample from G1 is shown in red . samples were collected simultaneously before initiation of treatment C ) Cumulative frequency of average density of fragment in CSF ( blue ) plasma ( red ) . vertical dashed represents 167

O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O 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) Fragment size distribution, determined by sWGS, in CSF (blue line), plasma (red dashed line) and urine (black dashed line) from patient G1. The 3 samples were collected simultaneously before initiation of treatment. B) Fragment size distribution of cfDNA, determined by sWGS, in CSF from 13 glioma patients. The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~145 bp and ~167 bp. The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red. All samples were collected simultaneously before initiation of treatment C) Cumulative frequency analysis of the average density of fragment size in CSF (blue) and plasma (red). The vertical dashed line represents 167 bp


Mean fragment density in with detected SCNAs in CSF ( yellow ) , those with no detected SCNAs ( blue )

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

Mean fragment size density in patients with detected SCNAs in the CSF (yellow), and those with no detected SCNAs (dark blue)


Log2 of difference in cfDNA fragment sizes between CSF samples with detected SCNAs those samples in which no SCNAs were

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

Log2 ratio of the difference in cfDNA fragment sizes between CSF samples with detected SCNAs and those samples in which no SCNAs were detected


amplitude of 10 bp periodic peaks in fragment is a signature for non - in CSF . amplitude of 10 bp periodic peaks in fragment distribution ( in range between 75 150 bp , see Methods ) when compared to mean copy number alteration calculated from sWGS revealed a significant negative correlation ( Pearson - 0 . 85 ; p = 0 . 0002 )

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

The amplitude of the 10 bp periodic peaks in fragment size is a signature for non-tumor DNA in the CSF. The amplitude of the 10 bp periodic peaks in fragment length distribution (in the range between 75 and 150 bp, see Methods) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation (Pearson -0.85; p=0.0002)


U2OS were transfected , synchronized at G1 / S - border by thymidine for 24 hours then either released into nocodazole for 16 hours ( unperturbed mitotic entry ) or released for 7 hours , treated with 0 . 5 μM Adriamycin for one hour after 16 hour G2 arrest were induced to recover by addition of caffeine ( checkpoint recovery ) . Mitotic index was scored on of Histone H3 - pS10 positive DAPI - nuclei normalized to untransfected controls . Black diamonds indicate siRNA - targeted genes from library , grey diamonds indicate positive ( Wip1 - depleted ) negative ( untransfected GAPDH - depleted ) controls grey diamonds indicate hits on two screens . Dotted lines indicate selection criteria for recovery - specific genes .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells were transfected, synchronized at the G1/S-border by thymidine for 24 hours and then either released into nocodazole for 16 hours (unperturbed mitotic entry) or released for 7 hours, treated with 0.5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine (checkpoint recovery). Mitotic index was scored based on the percentage of Histone H3-pS10 positive DAPI-nuclei and normalized to the untransfected controls. Black diamonds indicate individual siRNA-targeted genes from the library, light grey diamonds indicate positive (Wip1-depleted) and negative (untransfected and GAPDH-depleted) controls and dark grey diamonds indicate the hits based on the two screens. Dotted lines indicate selection criteria for recovery-specific genes.


A ) U2OS were transfected with four independent siRNA ' s from pools used in screen , treated as in figure 1 analyzed for mitotic index . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells were transfected with four independent siRNA's from the pools used in the screen, treated as in figure 1 and analyzed for mitotic index. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01)


B ) U2OS were synchronized with a single thymidine block , released into G2 damaged with 0 . 5 μM Adriamycin for one hour . After a 16 hour G2 arrest were harvested for western blot analyzed for Tlk2 .

O O O O O O O O O O O O O O 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-MEASURED_VAR O

B) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels.


C ) Live imaging of thymidine synchronized unperturbed or damaged G2 . Cumulative of entering mitosis were scored plotted .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

C) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted.


D ) U2OS were transfected with either a control siRNA or Tlk2 siRNA # 3 , synchronized damaged in G2 . were either harvested or treated with caffeine for 8 hours before harvest cycle distribution was analyzed by FACS . Percentages of in each quadrant are indicated .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

D) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized and damaged in G2. Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated.


E ) Tlk2Δ were generated using CRISPR / Cas9 genome editing . were synchronized in G2 by thymidine release , damaged with 0 . 5 μM Adriamycin for one hour . After a 16 - hour G2 arrest were induced to recover by addition of caffeine for 8 hours analyzed by FACS . Error bars represent SD , n = 4 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * * for P ≤ 0 . 001 ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release, damaged with 0.5 μM Adriamycin for one hour. After a 16-hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS. Error bars represent SD, n=4. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, *** for P≤0.001).


F ) U2TR stably expressing Tlk2 siRNA # 3 - insensitive tetracycline - inducible FLAG - Tlk2 - wt or FLAG - Tlk2 - D613A were thymidine synchronized damaged in G2 . Tetracycline was present form start of experiment where indicated .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

F) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt or FLAG-Tlk2-D613A were thymidine synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated.


G ) Caffeine induced recovery of lines shown in 2F . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 , * * * for P ≤ 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Caffeine induced recovery assay of cell lines shown in 2F. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01, *** for P≤0.001).


A ) U2OS were grown on glass coverslips synchronized in G2 after thymidine release . were fixed at indicated times after adriamycin - induced damage stained for DAPI γH2AX . Scale bar indicates 10 μm . B ) of in 3A . Each dot represents total signal of γH2AX in nucleus of a single . Bars indicate mean error bars indicate SD of data points in each condition . Whiskers represent 5 - 95 % of data points . Statistical significance was tested using an unpaired two - t - ( NS for P > 0 . 05 , * * * * for P ≤ 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release. Cells were fixed at the indicated times after adriamycin-induced DNA damage and stained for DAPI and γH2AX. Scale bar indicates 10 μm. B) Quantification of cells in 3A. Each dot represents the total signal of γH2AX in the nucleus of a single cell. Bars indicate the mean and error bars indicate the SD of the data points in each condition. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (NS for P>0.05, **** for P≤0.0001).


C ) U2OS were synchronized in G2 harvested at indicated times after induction of adriamycin - induced damage with or without Tlk2 - depletion . Samples were analyzed by western blotting for indicated .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

C) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin-induced DNA damage with or without Tlk2-depletion. Samples were analyzed by western blotting for the indicated proteins.


D ) U2OS were synchronized in G2 damaged with 6 Gy of IR . Samples were taken at indicated times after induction of damage with or without Tlk2 - depletion . Samples were analyzed by western blot for indicated . Band signal for Asf1A Asf1B was measured corrected for Ponceau S .

O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O

D) U2OS cells were synchronized in G2 and damaged with 6 Gy of IR. Samples were taken at the indicated times after induction of damage with or without Tlk2-depletion. Samples were analyzed by western blot for the indicated proteins. Band signal intensity for Asf1A and Asf1B was measured and corrected for Ponceau S staining.


E ) U2OS were transfected with indicated siRNA ' s treated as in 2A . Mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells were transfected with the indicated siRNA's and treated as in 2A. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).


F ) U2OS were synchronized with a single thymidine block , released into G2 damaged with 0 . 5 μM Adriamycin for one hour . After a 16 hour G2 arrest were harvested for western blot analyzed for indicated . Lanes were present on same blot but pasted together for comparison as indicated by lines .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels. Lanes were all present on the same blot but pasted together for comparison as indicated by the lines.


G ) were synchronized in S - or G2 - phase by a single thymidine block . Damage was induced by 0 . 5 μM Adriamycin for one hour were allowed to incubate for 16 hours afterwards . Samples were fixed at indicated cycle stages stained for Asf1A - pS166 . was measured relative to DAPI . Whiskers represent 5 - 95 % of data points .

O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

G) Cells were synchronized in S- or G2-phase by a single thymidine block. Damage was induced by 0.5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards. Samples were fixed at the indicated cell cycle stages and stained for Asf1A-pS166. Nuclear fluorescence intensity was measured relative to DAPI. Whiskers represent 5-95% of data points.


H ) were treated analyzed as in 2E . Induction of FLAG - Asf1A - 4D was induced using doxycycline . Error bars represent SD , n = 5 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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) Cells were treated and analyzed as in 2E. Induction of FLAG-Asf1A-4D was induced using doxycycline. Error bars represent SD, n=5. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).


I ) U2OS were synchronized in G2 by a single thymidine block where indicated treated with 0 . 5 μM Adriamycin for one hour after indicated time points were pre - extracted fixed . were stained for total of Histone H3 quantified relative to DAPI . Scale bar indicates 10 μm . Whiskers represent 5 - 95 % of data points .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

I) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0.5 μM Adriamycin for one hour and after the indicated time points were pre-extracted and fixed. Cells were stained for total levels of Histone H3 and quantified relative to DAPI. Scale bar indicates 10 μm. Whiskers represent 5-95% of data points.


J ) FLAG - Asf1A - 4D inducible were treated as in 3I expression was induced using doxycycline where indicated . Whiskers represent 5 - 95 % of data points . Statistical significance was tested using an unpaired two - t - ( * * * * for P ≤ 0 . 0001 ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FLAG-Asf1A-4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (**** for P≤0.0001).


A ) U2OS were treated as in 2B after which mRNA was isolated analyzed by qPCR . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells were treated as in 2B after which mRNA was isolated and analyzed by qPCR. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).


B ) Tlk2Δ 9 . 3 . 1 were treated analyzed as in 3A . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tlk2Δ 9.3.1 cells were treated and analyzed as in 3A. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01).


C ) U2OS were synchronized in G2 harvested or synchronized in G2 harvested 16 hours after adriamycin - induced damage . Samples were analyzed by western blot for indicated .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

C) U2OS cells were synchronized in G2 and harvested or synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.


D ) U2OS were transfected with indicated siRNA ' s , synchronized in G2 damaged with 6 Gy of IR . were harvested 24 hours later mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells were transfected with the indicated siRNA's, synchronized in G2 and damaged with 6 Gy of IR. Cells were harvested 24 hours later and the mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).


F ) U2OS were transfected with indicated siRNA ' s , arrested in G2 with Adriamycin induced to recover by addition of caffeine . Mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS cells were transfected with the indicated siRNA's, arrested in G2 with Adriamycin and induced to recover by addition of caffeine. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).


G ) U2OS were synchronized in G2 harvested 16 hours after adriamycin - induced damage . Samples were analyzed by western blot for indicated .

O 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) U2OS cells were synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.


H ) U2TR stably expressing Tlk2 siRNA # 3 - insensitive tetracycline - inducible FLAG - Tlk2 - wt were thymidine synchronized , damaged in G2 harvested 24 hours afterwards . Tetracycline was either absent ( off ) , added from start of experiment ( early ) or for 16 hours after induction of damage ( late ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt were thymidine synchronized, damaged in G2 and harvested 24 hours afterwards. Tetracycline was either absent (off), added from the start of the experiment (early) or for 16 hours after induction of DNA damage (late).


I ) were treated as in 5G recovery was induced by addition of caffeine for 8 hours . Mitotic index was analyzed by FACS normalized to control . Error bars represent SD of three independent experiments . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - t - ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were treated as in 5G and recovery was induced by addition of caffeine for 8 hours. Mitotic index was analyzed by FACS and normalized to the control. Error bars represent SD of three independent experiments. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).


B . Left , representative of Western blot for ARID1A expression in lysates extracted from cortex of Arid1afl / + ( WT ) , cHet , cKO mice using indicated antibodies . Right , of normalized to β - actin ( n = 3 mice per group ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O 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. Left, representative images of Western blot for ARID1A expression in lysates extracted from the cortex of Arid1afl/+(WT), cHet, and cKO mice using the indicated antibodies. Right, quantification of protein levels normalized to β-actin (n = 3 mice per group).


C . Left , representative of Western blot for ARID1A expression in lysates extracted from hippocampi of Arid1afl / + , cHet , cKO mice . Right , of which were normalized to β - actin ( n = 4 mice per group ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O 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. Left, representative images of Western blot for ARID1A expression in lysates extracted from the hippocampi of Arid1afl/+, cHet, and cKO mice. Right, quantification of protein levels which were normalized to β-actin (n = 4 mice per group).


D . Immunostaining against ARID1A ( red ) in dentate gyrus ( DG ) , cortex , CA1 , CA3 regions of Arid1afl / + cHet mice .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

D. Immunostaining against ARID1A (red) in the dentate gyrus (DG), cortex, CA1, and CA3 regions of Arid1afl/+and cHet mice.


E . cHet mice spent more time reaching platform during 5 - day training in Morris water maze compared to control group mice . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

E. cHet mice spent more time reaching the platform during 5-day training in Morris water maze assay compared to the control group mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


F . In probe trial , cHet mice displayed spatial learning disabilities compared with Arid1afl / + mice . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

F. In the probe trial, cHet mice displayed spatial learning disabilities compared with Arid1afl/+ mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


G . cHet mice undertook fewer platform crossings than Arid1afl / + mice in Morris water maze . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

G. cHet mice undertook fewer platform crossings than Arid1afl/+ mice in the Morris water maze assay. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


H - J . cHet mice were evaluated using Barnes maze . During training phase , cHet mice ( n = 12 mice ) failed to diminish latency of first entrance to hiding box ( H ) , indicating that Arid1a haploinsufficiency results in impaired spatial memory . Probe trials demonstrated that cHet mice also had spatial memory deficits ( I ) , as they visited target hole less often than Arid1afl / + mice ( n = 12 mice ) ( J ) .

O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O

H-J. cHet mice were evaluated using the Barnes maze test. During the training phase, the cHet mice (n = 12 mice) failed to diminish the latency of the first entrance to the hiding box (H), indicating that Arid1a haploinsufficiency results in impaired spatial memory. Probe trials demonstrated that cHet mice also had spatial memory deficits (I), as they visited the target hole less often than the Arid1afl/+ mice (n = 12 mice) (J).


A . Representative traces of spontaneous miniature excitatory postsynaptic currents ( mEPSCs ) in CA1 hippocampal acute slices from Arid1afl / + cHet mice . B . of amplitude ( B , left ) frequency ( B , right ) spontaneous miniature excitatory postsynaptic currents ( mEPSCs ) in Arid1afl / + cHet hippocampal CA1 neurons . Arid1afl / + ( 17 neurons from n = 3 mice ) cHet ( 15 neurons from n = 4 mice ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A. Representative traces of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in CA1 hippocampal acute slices from Arid1afl/+ and cHet mice. B. Quantification of amplitude (B, left) and frequency (B, right) spontaneous miniature excitatory postsynaptic currents (mEPSCs) in Arid1afl/+ and cHet hippocampal CA1 neurons. Arid1afl/+ (17 neurons from n =3 mice) and cHet (15 neurons from n = 4 mice).


C . Representative recording of paired - at 50 - ms interpulse interval ms from slices prepared from Arid1afl / + ( n = 4 ) cHet ( n = 4 ) mice . D . Paired - facilitation ( PPF ) studies across different interpulse intervals ( 20 ms , 40 ms , 50 ms , 100 ms , 200 ms , 300ms , 500 ms ) revealed a significant difference in paired - ratios at 50 - ms intervals .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative recording of the paired-pulse ratio at the 50-ms interpulse interval ms from slices prepared from Arid1afl/+ (n = 4) and cHet (n = 4) mice. D. Paired-pulse facilitation (PPF) studies across different interpulse intervals (20 ms, 40 ms, 50 ms, 100 ms, 200 ms, 300ms, and 500 ms) revealed a significant difference in paired-pulse ratios at 50-ms intervals.


E . Summary plots showing time course of long - term potentiation ( LTP ) induced by frequency stimulation in CA1 region from Arid1afl / + or cHet mice . Field excitatory postsynaptic potential ( fEPSP ) traces before ( black ) after ( black or red ) are shown in inset above . F . Average amplitude of LTP measured at 55 - 60 min postinduction ( n = 8 slices from 4 mice per group ; 10 fEPSP slope ( % ) values were collected from 1 slice ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Summary plots showing the time course of long-term potentiation (LTP) induced by frequency stimulation in the CA1 region from Arid1afl/+or cHet mice. Field excitatory postsynaptic potential (fEPSP) traces before (black) and after (black or red) are shown in the inset above. F. Average amplitude of LTP measured at 55-60 min postinduction (n = 8 slices from 4 mice per group; 10 fEPSP slope (%) values were collected from 1 slice).


G . Representative Western blot for expression of several LTP - related in Arid1afl / + cHet hippocampi . H . of expression of each normalized to β - actin level .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

G. Representative Western blot images for the expression of several LTP-related proteins in the Arid1afl/+ and cHet hippocampi. H. Quantification of the expression of each protein normalized to the β-actin level.


A . Golgi - stained coronal section in hippocampal CA1 region of 2 - month - old Arid1afl / + cHet mice . Scale bar , 100µm . B - D . Partial loss of Arid1a significantly decreased dendritic ( B ) , dendritic nodes ( C ) , dendritic ends ( D ) in cHet neurons .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

A. Golgi-stained coronal section in the hippocampal CA1 region of 2-month-old Arid1afl/+ and cHet mice. Scale bar, 100µm. B-D. Partial loss of Arid1a significantly decreased dendritic length (B), dendritic nodes (C), and dendritic ends (D) in cHet neurons. E


A . Left , Western blot of H3K27ac in forebrain tissues of Arid1afl / + cHet mice . Right , relative quantitation of of H3K27ac normalized to H3 level ( n = 3 mice per group ) .

O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O

A. Left, Western blot analysis of H3K27ac in forebrain tissues of Arid1afl/+and cHet mice. Right, relative quantitation of the intensity of H3K27ac normalized to the H3 level (n=3 mice per group).


B . Left , Western blot of H3K27ac in forebrain tissues of vehicle - or acetate - treated cHet mice . Right , relative quantitation of of H3K27ac which was normalized to H3 level ( n = 4 mice per group ) .

O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O

B. Left, Western blot analysis of H3K27ac in forebrain tissues of vehicle- or acetate-treated cHet mice. Right, relative quantitation of the intensity of H3K27ac which was normalized to the H3 level (n=4 mice per group).


D . Immunofluorescence ( IF ) of H3K27ac ( green ) Vgult1 ( red ) in forebrain tissues of Arid1afl / + cHet mice , respectively . IF was performed on 40 - μm thick floating sections . Relative intensities of H3K27ac decreased upon haploinsufficiency of Arid1a in cortex . Scale Bar , 20μm . Arid1afl / + ( n = 7 ) cHet ( n = 4 ) mice .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

D. Immunofluorescence (IF) staining of H3K27ac(green) and Vgult1(red) in forebrain tissues of Arid1afl/+ and cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac decreased upon the haploinsufficiency of Arid1a in the cortex. Scale Bar, 20μm. Arid1afl/+ (n = 7) and cHet (n = 4) mice.


E . Immunofluorescence of H3K27ac ( green ) Vgult1 ( red ) in cortex tissues of vehicle - or acetate - treated cHet mice , respectively . IF was performed on 40 - μm thick floating sections . Relative intensities of H3K27ac increased after treatment with acetate in cortex of cHet mice . Scale Bar , 20μm ( n = 3 mice per group ) .

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

E. Immunofluorescence staining of H3K27ac (green) and Vgult1 (red) in cortex tissues of vehicle- or acetate-treated cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac increased after treatment with acetate in the cortex of cHet mice. Scale Bar, 20μm (n=3 mice per group).


F - H . Morris water maze of Arid1afl / + cHet mice treated with vehicle or acetate . Acquisition task , latency to find platform was used to assess learning ability . Latency to platform was measured during training trials ( F ) . Latency to platform was measured during probe ( G ) . ( H ) Retention task , times in target crossings were used to assess memory retention . times in target crossings were measured in probe , n = 10 - 15 mice per group .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H. Morris water maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. Acquisition task, the latency to find the platform was used to assess learning ability. Latency to the platform was measured during training trials (F). Latency to the platform was measured during probe test (G). (H) Retention task, the times in the target crossings were used to assess memory retention. The times in the target crossings were measured in probe tests, n = 10-15 mice per group.


I - K . Barnes maze of Arid1afl / + cHet mice treated with vehicle or acetate . ( I ) During training phase , time to find target in Arid1afl / + cHet mice that were treated with vehicle or acetate for 28 days . ( J ) In probe trials , time of latency to platform to find target in Arid1afl / + cHet mice treated with vehicle or acetate . ( K ) In probe trials , numbers of target crossings of Arid1afl / + cHet mice treated with vehicle or acetate ( n = 12 mice )

O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

I-K. Barnes maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. (I) During the training phase, the time to find the target in Arid1afl/+ and cHet mice that were treated with vehicle or acetate for 28 days. (J) In probe test trials, the time of latency to the platform to find the target in Arid1afl/+ and cHet mice treated with vehicle or acetate. (K) In probe trials, the numbers of target crossings of Arid1afl/+ and cHet mice treated with vehicle or acetate (n = 12 mice)


E . Representative of secondary branches of apical dendrites of vehicle - or acetate - treated cHet CA1 pyramidal neurons stained by Golgi - Cox method ( at least 20 neurons from 4 mice for each group ) . Scale bar , 5 μm . F . Spine density per 10 μm was measured on secondary dendrites in ( E ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of secondary branches of apical dendrites of vehicle- or acetate-treated cHet CA1 pyramidal neurons stained by the Golgi-Cox method (at least 20 neurons from 4 mice for each group). Scale bar, 5 μm. F. Spine density per 10 μm was measured on secondary dendrites in (E).


A . Representative image of vehicle - acetate - treated neurons derived from WT or ARID1A KO hESCs on day 40 of neural differentiation Scale Bar , 20μm B . of total neurite of hESC - derived neurons after treatment with acetate or vehicle . n = at least 34 neurons for each group from 3 independent experiments .

O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

A. Representative image of vehicle- and acetate-treated neurons derived from WT or ARID1A KO hESCs on day 40 of neural differentiation Scale Bar, 20μm B. Quantification of total neurite length of hESC-derived neurons after treatment with acetate or vehicle. n = at least 34 neurons for each group from 3 independent experiments. C


D . Representative of dendrites ( MAP2 , green ) showing localization of foci of pre - postsynaptic complexes , synaptophysin ( red ) , PSD - 95 ( ) in control , acetate treatment of WT ARID1A KO hESC - derived neurons on day 55 of neural differentiation . Scale Bar , 5μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

D. Representative images of dendrites (MAP2, green) showing localization of foci of the pre- and postsynaptic protein complexes, synaptophysin (red), and PSD-95 (white) protein in control, acetate treatment of WT and ARID1A KO hESC-derived neurons on day 55 of neural differentiation. Scale Bar, 5μm.


E , F . of spine numbers from synaptophysin ( E ) PSD - 95 ( F ) stained secondary dendrites of neurons on day 55 of neural differentiation ( Data are representative of three independent experiments . )

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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. Quantification of the spine numbers from synaptophysin (E) and PSD-95 protein (F) stained secondary dendrites of neurons on day 55 of neural differentiation (Data are representative of three independent experiments.)


C Genomic PCR indicating existence of MpFGMYB in both [ Y ] [ X ] genomes of M . polymorpha . Two biological replicates were analyzed . Constitutively expressed MpEF1α was used as a control .

O O O O O O B-MEASURED_VAR O O O O O 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 Genomic PCR analysis indicating the existence of MpFGMYB in both male [Y] and female [X] genomes of M. polymorpha. Two biological replicates were analyzed. Constitutively expressed MpEF1α was used as a control.


D Real - time RT - PCR analyses indicating preferential accumulation of MpFGMYB transcripts in sexual organs sporophytes . MpEF1α was used for normalization . Measurements of six biological replicates for thalli sporophyte , three biological replicates for gametangiophore are plotted . Bars represent mean ± SD . Symbols above bars indicate grouping by p < 0 . 05 in a Tukey - Kramer .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Real-time RT-PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes. MpEF1α was used for normalization. Measurements of six biological replicates for thalli and sporophyte, and three biological replicates for gametangiophore are plotted. Bars represent mean ± SD. Symbols above the bars indicate grouping by p <0.05 in a Tukey-Kramer test.


E A transcriptional reporter with 5 ′ - 3 ′ - flanking sequences revealed transcription of MpFGMYB throughout mature archegonia . Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

E A transcriptional reporter with 5′- and 3′-flanking sequences revealed transcription of MpFGMYB throughout mature archegonia. Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.


F MpFGMYB - Citrine fusion expressed using 5 ′ - 3 ′ - flanking sequences rescued Mpfgmybge - 1 mutant accumulated in nuclei of egg ventral canal ( VCC ) Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine .

O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O 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 MpFGMYB-Citrine fusion proteins expressed using the 5′- and 3′-flanking sequences rescued the Mpfgmybge-1 mutant and accumulated in the nuclei of the egg and the ventral canal cell (VCC) Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.


G A transcriptional AtMYB64 reporter ( AtMYB64 - NYG ) is specifically expressed in four types of A . thaliana sac Scale bar : 25 µm . Green , YFP ; , walls .

O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O 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 A transcriptional AtMYB64 reporter (AtMYB64-NYG) is specifically expressed in all four cell types of the A. thaliana embryo sac Scale bar: 25 µm. Green, YFP fluorescence; white, cell walls.


H Expression of AtMYB64 - Citrine fusion under AtMYB64 promoter was detected in central ( CC ) egg ( EC ) of A . thaliana sac Scale bar : 25 µm . Green , Citrine ; , walls .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O 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 Expression of AtMYB64-Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells (CC) and egg cells (EC) of the A. thaliana embryo sac Scale bar: 25 µm. Green, Citrine fluorescence; white, cell walls.


B Diagnosis of genetic sex using Y - X - chromosome - linked rbm27 rhf73 markers , respectively . Two biological replicates were analyzed for each genotype .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O

B Diagnosis of genetic sex using Y- and X-chromosome-linked rbm27 and rhf73 markers, respectively. Two biological replicates were analyzed for each genotype.


C - L Gametangiophore ( C - G ) gamete development ( H - L ) of wild - type mutant . Scale bars , 5 mm ( C - G ) , 10 µm ( H J ) , 100 µm ( I , K , L ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L Gametangiophore morphology (C-G) and gamete development (H-L) of wild-type and mutant plants. Scale bars, 5 mm (C-G), 10 µm (H and J), 100 µm (I, K, and L).


A - C DAPI - visualization of sperm formation in wild - type ( A ) , Mpfgmyb [ X ] ( B ) , Mpfgmyb [ Y ] ( C ) . Note that background DAPI visualizes flagella ( arrows ) in addition to nuclei ( arrowheads ) . ( B ′ ) is an enlarged image of boxed region in ( B ) , visualizing an incompletely condensed nucleus . Scale bars , 5 µm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bars, 5 µm.


D RT - PCR indicating acquisition of - like autosomal gene expression patterns in Mpfgmyb [ X ] antheridiophores . Two independent Mpfgmyb [ X ] mutant alleles were analyzed .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O

D RT-PCR analysis indicating acquisition of male-like autosomal gene expression patterns in Mpfgmyb [X] antheridiophores. Two independent Mpfgmyb [X] mutant alleles were analyzed.


E , F analyses visualizing abnormal arrangement of axonemal microtubules in Mpfgmyb [ X ] sperm ( F ) , as compared with those of wild - type ( E ) . Scale bars , 100 nm .

O O O O O O O O O O O B-CONTROLLED_VAR O O 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 TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [X] sperm (F), as compared with those of wild-type males (E). Scale bars, 100 nm.


A - seq showing - specific accumulation of lncRNAs derived from MpFGMYB 3 ′ region . Diagrams illustrate wild - type mutant MpFGMYB / SUF loci . Folded lines with a ∆ symbol indicate a deletion .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

A RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region. Diagrams illustrate wild-type and mutant MpFGMYB/SUF loci. Folded lines with a ∆ symbol indicate a deletion.


B RT - PCR of wild - type genetically suf mutants revealed loss of SUF expression gain of MpFGMYB expression after induction of reproductive growth by far - red irradiation . Two independent suf mutant alleles were analyzed . SUF primer pair used here flanked an intron duplicated bands of SUF likely represent spliced unspliced forms .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O

B RT-PCR analysis of wild-type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far-red irradiation. Two independent suf mutant alleles were analyzed. The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms.


C - H Gametangiophore ( C , E G ) gametangium development ( D , F H ) of with designated genotypes . Scale bars , 1 mm ( C , E G ) , 20 µm ( D ) , 50 µm ( F ) , 100 µm ( H ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Gametangiophore morphology (C, E and G) and gametangium development (D, F and H) of plants with the designated genotypes. Scale bars, 1 mm (C, E and G), 20 µm (D), 50 µm (F), 100 µm (H).


A gametangiophore of MpEF1pro : SUF / suf - 30ge [ Y ] , indicating inability of transgenic SUF overexpression in rescuing feminization phenotype of sufge [ Y ] . Scale bar , 2mm .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O

A gametangiophore of MpEF1pro:SUF/suf-30ge [Y], indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [Y]. Scale bar, 2mm.


B Real - time RT - PCR analyses confirming SUF transcript accumulation in SUF - overexpressing lines . Constitutively expressed MpEF1α was used as control . Measurements of six biological replicates for WT [ Y ] , three biological replicates for each SUF - overexpressing are plotted . Data information : bars represent mean ± SD . Asterisks indicate significant differences from WT [ Y ] ( B ) ( p < 0 . 05 , two - Student ' s t - ) .

O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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 Real-time RT-PCR analyses confirming SUF transcript accumulation in SUF-overexpressing lines. Constitutively expressed MpEF1α was used as control. Measurements of six biological replicates for WT [Y], and three biological replicates for each SUF-overexpressing line are plotted. Data information: bars represent mean ± SD. Asterisks indicate significant differences from WT [Y] (B) (p <0.05, two-tailed Student's t-test).


D Real - time RT - PCR measurement of MpFGMYB transcript . Three biological replicates are analyzed for each . Data information bars represent mean ± SD . Asterisks indicate significant differences from WT [ X ] ( D ) ( p < 0 . 05 , two - Student ' s t - ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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 Real-time RT-PCR measurement of MpFGMYB transcript levels. Three biological replicates are analyzed for each line. Data information bars represent mean ± SD. Asterisks indicate significant differences from WT [X] (D) (p <0.05, two-tailed Student's t-test).


E - H Gametangiophores ( E , G ) gametangia ( F , H ) of gMpFGMYB - S [ Y ] ( E , F ) gMpFGMYB - L [ Y ] ( G , H ) . Scale bars , 1 mm ( E , G ) , 50 μm ( F ) , 100 μm ( H ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O 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-H Gametangiophores (E, G) and gametangia (F, H) of gMpFGMYB-S [Y] (E, F) and gMpFGMYB-L [Y] (G, H). Scale bars, 1 mm (E, G), 50 μm (F), 100 μm (H).


A ) Transmission electron microscopy ( ) of MoDCs infected with P . gingivalis for 2 , 12 24 hours ( left , middle right panels ) . sections show intra - extra - contents of Cont . ( un - infected ) , Pg381 , Mfa1 + Pg FimA + Pg infected MoDCs for different time

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O

A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points


B ) figure shows intracellular content of P . gingivalis strains after 24 hours of infection . MoDCs infected with Pg381 or isogenic mutants strains were lysed survived intracellular were re - suspended maintained in anaerobic broth for 5 days . data represents CFU within MoDCs harvested from three healthy individuals . means ± standard deviation ( in triplicates ) were analyzed by One - way ANOVA of different groups Tukey ' s for multiple group comparisons within 3 different experiments ( * statistical significance at p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey's test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001).


A ) DC - SIGN mRNA expression in P . gingivalis - infected MoDCs at 0 . 1 , 1 10 MOIs . figure shows gene expression after 12 hours of Pg381 mutant strains infections . target gene ( DC - SIGN ) was normalized using endogenous control GAPDH ( ΔCt ) fold regulations were calculated using 2 - ( ΔΔCt ) method . statistical was performed using t - , which accounts for clustering of infected un - infected controls within 3 different experiments ( * p < 0 . 001 ) .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001).


B ) Immuno - electron microscopy of un - infected MoDCs ( Cont . ) ( upper panel ) , MoDCs infected with Pg381 ( middle panel ) Mfa1 + Pg mutants ( lower panel ) . Gold particles ( marked with red rings ) for positive DC - SIGN were detected in cytoplasm of infected with Mfa1 + Pg strains . Minimal positive for DC - SIGN was detected in membranes of MoDCs infected with Pg381 , while no gold labeling was detected in these .

O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells.


C ) Flow cytometry of surface DC - SIGN in human MoDCs after infection with Pg381 , Mfa1 + Pg FimA + Pg . of used Kruskal - Wallis of different groups Dunn ' s for multiple comparisons 3 different experiments ( * p < 0 . 01 ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O 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) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons 3 different experiments (* p<0.01).


A ) Pg381 mutant strains after 24 hr incubation with human MoDCs with / without Rapamycin treatment . survived were measured after maintaining lysed MoDCs suspension in anaerobic broth for 5 days . plot represents means ± standard deviation of CFU within MoDCs harvested from three healthy individuals ( * P < 0 . 001 ) . of readings used One - way AVOVA of different groups Tukey ' s for multiple comparisons .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey's test for multiple comparisons.


B ) Epifluorescence microscopy of MoDCs treated with Rapamycin 1 hour after P . gingivalis infections . LC3 - II ( red - dye ) strains ( green CFSE ) were studied in MoDCs 11 hours after Rapamycin treatment ( 12 hours after infections ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections).


C ) Quantifications of of CFSE - labeled P . gingivalis LC3 - II signals within infected MoDCs using NIS - Elements BR software . One - way ANOVA was used to compare means of of different groups Tukey ' s for multiple comparisons ( * P < 0 . 001 ) .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O 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) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons (* P<0.001).


A ) Survival of P . gingivalis strains within MoDCs after 6 , 12 , 24 48 hours . Blue lines show P . gingivalis strains survival within MoDCs their survival in anaerobic condition in absence of DCs are showed in black lines .

O O O O O O O O O O O O O O O O O O O O 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) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines.


effect of rapamycin on Mfa1 + Pg , Pg381 FimA + Pg survival within MoDCs are shown in figures B , C D respectively . A three - factor repeated measures ANOVA using mixed models was used to effect of rapamycin treatment over time on OD reading . survival curves for strains are showed in blue , while effect of rapamycin treatments are in red . survivals in absence of MoDCs with without rapamycin are plotted in grey black , respectively . Statistical showed that by rapamycin treatment overtime interaction indicates pattern of means in each ( Mfa1 + Pg , Pg381 FimA + Pg ) between treated ( rapamycin ) untreated were significantly different overtime ( p - value < 0 . = 001 ) .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).


A ) Epifluorescence microscopy of MoDCs infected with Pg381 , Mfa1 + Pg , FimA + Pg MFB strains after 12 hours . LC3 - II was detected in red - ( Texas red ) dye strains were pre - labeled with CFSE ( green ) . Co - localization of P . gingivalis LC3 - II showed in right panels .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O

A) Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1+Pg, FimA+Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of P. gingivalis and LC3-II showed in the right panels.


B ) Quantifications of of LC3 - II within infected MoDCs using NIS - Elements BR software . One - way ANOVA was used to compare means of of different groups Tukey ' s for multiple comparisons of three different experiments ( * p < 0 . 001 , # p < 0 . 01 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons of three different experiments (* p<0.001, # p<0.01).


C ) uptake by MoDCs was determined by CFSE ( * p < 0 . 001 , # p < 0 . 01 ) .

O 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) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* p<0.001, # p<0.01).


A ) Immuno - of LC3 - II P . gingivalis within MoDCs pre - treated with GP120 ( DC - SIGN blocker ) after 12 hours of infection . LC3 - II was detected in red - dye strains were pre - labeled with green CFSE .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.


B ) of LC3 - II P . gingivalis strains signal ( in triplicates ) were statistically analyzed ( * # p < 0 . 001 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001).