B . Heatmaps representing different monocyte clusters identified by FlowSOM , with percentages in healthy donors ( CTR ) and COVID - 19 patients . The colors in the heat map represent the median of the arcsinh , 0 - 1 transformed marker expression calculated over cells from all the samples , varying from blue for lower expression to red for higher expression . Each cluster has a unique color assigned ( bar on the left which colors correspond to those used in panel A ) .

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B. Heatmaps representing different monocyte clusters identified by FlowSOM, with percentages in healthy donors (CTR) and COVID-19 patients. The colors in the heat map represent the median of the arcsinh, 0-1 transformed marker expression calculated over cells from all the samples, varying from blue for lower expression to red for higher expression. Each cluster has a unique color assigned (bar on the left which colors correspond to those used in panel A).


C . Differential analysis between healthy donors ( CTR , n = 8 ) and COVID - 19 ( COVID , n = 7 ) . The heatmap represents arcsine - square - root transformed cell frequencies that were subsequently normalized per cluster ( rows ) to mean of zero and standard deviation of one . The color of the heat varies from blue , indicating relative under - representation , to red indicating relative over - representation . Bar and numbers at the right indicate significant differentially abundant clusters ( green ) and adjusted p values ( Differential analysis performed by diffcyt package present in the Catalyst package ) . The statistical analyses was done using GLMM ( according to the actual guidelines ) and all p - values were Bonferroni corrected . Clusters were sorted according to the adjusted p values , so that the cluster at the top shows the most significant abundance changes between the two conditions . D . Boxes and whiskers plots representing maximum , minimum , median value , 25 and 75 percentile of different subpopulations of monocytes in healthy donors ( CTR , n = 8 ) and COVID - 19 patients ( COVID , n = 7 ) .

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C. Differential analysis between healthy donors (CTR, n=8) and COVID-19 (COVID, n=7). The heatmap represents arcsine-square-root transformed cell frequencies that were subsequently normalized per cluster (rows) to mean of zero and standard deviation of one. The color of the heat varies from blue, indicating relative under-representation, to red indicating relative over-representation. Bar and numbers at the right indicate significant differentially abundant clusters (green) and adjusted p values (Differential analysis performed by diffcyt package present in the Catalyst package). The statistical analyses was done using GLMM (according to the actual guidelines) and all p-values were Bonferroni corrected. Clusters were sorted according to the adjusted p values, so that the cluster at the top shows the most significant abundance changes between the two conditions. D. Boxes and whiskers plots representing maximum, minimum, median value, 25 and 75 percentile of different subpopulations of monocytes in healthy donors (CTR, n=8) and COVID-19 patients (COVID, n=7).


E . Scatter plots showing the frequency of PD - 1 + cells , PD - L1 + cells among total monocytes in healthy controls ( CTR , n = 8 ) and COVID - 19 patients ( COVID , n = 7 ) . A scatter plot reporting the median fluorescence intensity ( MFI ) of PD - L1 in total monocytes is also shown . Data represent individual values , mean ± standard error of the mean . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

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E. Scatter plots showing the frequency of PD-1+ cells, PD-L1+ cells among total monocytes in healthy controls (CTR, n=8) and COVID-19 patients (COVID, n=7). A scatter plot reporting the median fluorescence intensity (MFI) of PD-L1 in total monocytes is also shown. Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


A . Quantification of cytokines and other mediators in plasma obtained from COVID - 19 patients ( COVID , n = 15 ) and healthy controls ( CTR , n = 15 ) . Data represent individual values , mean ± standard error of the mean . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

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A. Quantification of cytokines and other mediators in plasma obtained from COVID-19 patients (COVID, n=15) and healthy controls (CTR, n=15). Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


B . Representative dot plots showing percentages of immature and mature circulating monocytes in CTR and COVID - 19 patients . Scatter plots showing the quantification of immature and mature monocytes in healthy controls ( CTR , n = 6 ) and COVID - 19 patients ( COVID , n = 7 ) are reported . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

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B. Representative dot plots showing percentages of immature and mature circulating monocytes in CTR and COVID-19 patients. Scatter plots showing the quantification of immature and mature monocytes in healthy controls (CTR, n=6) and COVID-19 patients (COVID, n=7) are reported. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


( A ) Bar chart showing the relative abundance ( signal intensity ) of the phosphorylated tau species identified using an exploratory IP - MS approach using HT7 antibody on TBS - soluble control and Alzheimer ' s disease ( AD ) pools . Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP - MS : mono - phosphorylated p - tau231 and double phosphorylated p - tau ( 231 + 235 ) .

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(A) Bar chart showing the relative abundance (signal intensity) of the phosphorylated tau species identified using an exploratory IP-MS approach using HT7 antibody on TBS-soluble control and Alzheimer's disease (AD) pools. Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP-MS: mono-phosphorylated p-tau231 and double phosphorylated p-tau(231+235).


( A ) Box - and - whiskers plot showing the levels of CSF p - tau235 in neurological controls ( n = 21 ) and biologically defined Alzheimer ' s disease ( AD ) cases ( n = 19 ) .

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(A) Box-and-whiskers plot showing the levels of CSF p-tau235 in neurological controls (n=21) and biologically defined Alzheimer's disease (AD) cases (n=19).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in the different groups : amyloid - negative cognitively unimpaired ( CU - ) participants ( n = 50 ) , participants across the Alzheimer ' s disease ( AD ) continuum ( amyloid - positive cognitively unimpaired ( CU + , n = 32 ) ; amyloid - positive mild cognitive impairment ( MCI + , n = 20 ) ; AD ( n = 20 ) and non - AD cases ( n = 19 ) . CSF p - tau235 is highly specific for AD pathology . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

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(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in the different groups: amyloid-negative cognitively unimpaired (CU-) participants (n=50), participants across the Alzheimer's disease (AD) continuum (amyloid-positive cognitively unimpaired (CU+, n=32); amyloid-positive mild cognitive impairment (MCI+, n=20); AD (n=20) and non-AD cases (n=19). CSF p-tau235 is highly specific for AD pathology. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in A - T - participants and across preclinical Alzheimer ' s disease ( AD , dichotomised as A + T - and A + T + ) . CSF p - tau235 was increased already in A + T - , which represents the early cases within preclinical AD . A prominent increase was observed from A + T - to A + T + cases , the latter representing late preclinical AD cases . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

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(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in A-T- participants and across preclinical Alzheimer's disease (AD, dichotomised as A+T- and A+T+). CSF p-tau235 was increased already in A+T-, which represents the early cases within preclinical AD. A prominent increase was observed from A+T- to A+T+ cases, the latter representing late preclinical AD cases. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method ( Loess curve ) . Changes on biomarkers levels in CSF are represented as z - scores using Aβ PET in centiloid scale ( CL ) as a proxy of disease progression . Abnormal biomarker levels were determined as two standard deviations ( SD ) above the mean . Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 ( Mila - Aloma et al . , 2020 ) . All samples were run in singlicates .

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Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method (Loess curve). Changes on biomarkers levels in CSF are represented as z-scores using Aβ PET in centiloid scale (CL) as a proxy of disease progression. Abnormal biomarker levels were determined as two standard deviations (SD) above the mean. Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 (Mila-Aloma et al., 2020). All samples were run in singlicates.


( A ) Stacked bar chart depicting the percentages of [ 231 + / 235 + ] , [ 231 + / 235 - ] , [ 231 - / 235 - ] and [ 231 - / 235 + ] cases in each preclinical group . Table below indicates the exact percentages of participants in each group ( exact number of participants in parenthesis ) . Discordant cases with the sequential phosphorylation hypothesis [ 231 - / + 235 ] shown in grey .

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(A) Stacked bar chart depicting the percentages of [231+/235+], [231+/235-], [231-/235-] and [231-/235+] cases in each preclinical group. Table below indicates the exact percentages of participants in each group (exact number of participants in parenthesis). Discordant cases with the sequential phosphorylation hypothesis [231-/+235] shown in grey.


( B ) Correlation between CSF p - tau235 and p - tau231 in the whole ALFA + cohort ( Spearman ' s rank correlation : rS = 0 . 80 , P ˂ 0 . 0001 ) . Assay cut - offs were determined as the mean + 2 SD of the A - T - group ( defining p - tau231 and p - tau235 positivity or negativity in each participant ) . Cut - off values are indicated in red ( 19 . 92 and 9 . 59 pg / mL for CSF p - tau235 and p - tau231 respectively ) and displayed with black dashed lines , resulting in four quadrants , each of them representing the four different positive or negatively status for each biomarker . Discordant cases with the sequential phosphorylation hypothesis ( [ 231 - / + 235 ] ) showed on the lower right quadrant ( 2 . 35 % of the total , 9 participants out 383 ) . All samples were run in singlicates .

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(B) Correlation between CSF p-tau235 and p-tau231 in the whole ALFA+ cohort (Spearman's rank correlation: rS = 0.80, P˂0.0001). Assay cut-offs were determined as the mean + 2 SD of the A-T- group (defining p-tau231 and p-tau235 positivity or negativity in each participant). Cut-off values are indicated in red (19.92 and 9.59 pg/mL for CSF p-tau235 and p-tau231 respectively) and displayed with black dashed lines, resulting in four quadrants, each of them representing the four different positive or negatively status for each biomarker. Discordant cases with the sequential phosphorylation hypothesis ([231-/+235]) showed on the lower right quadrant (2.35% of the total, 9 participants out 383). All samples were run in singlicates.


( A , B ) Two - dimensional tryptic mapping of YFP - p27 , YFP - p27T198A and a mixture of both ( A ) . Phosphoamino - acid analysis of spots a and b ( B ) . The migration of phosphoserine ( S ) , phosphothreonine ( T ) and phosphotyrosine ( Y ) is marked with circles in B . The asterisk indicates the origin of migration .

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(A, B) Two-dimensional tryptic mapping of YFP-p27, YFP-p27T198A and a mixture of both (A). Phosphoamino-acid analysis of spots a and b (B). The migration of phosphoserine (S), phosphothreonine (T) and phosphotyrosine (Y) is marked with circles in B. The asterisk indicates the origin of migration.


( C - E ) MCF - 7 transfected with YFP - p27 or YFP - p27T198A mutant ( C , D ) or cotransfected with YFP - p27 and an empty RFP vector in a 5 : 1 ( YFP - p27 : RFP ) ratio ( E ) were treated with ( + ) or without ( − ) MG132 ( 10 μM ) for 12 h before western blot analysis ( C ) and fluorescence microscopy ( D , E ) .

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(C-E) MCF-7 transfected with YFP-p27 or YFP-p27T198A mutant (C, D) or cotransfected with YFP-p27 and an empty RFP vector in a 5:1 (YFP-p27: RFP) ratio (E) were treated with (+) or without (−) MG132 (10 μM) for 12 h before western blot analysis (C) and fluorescence microscopy (D, E).


( F ) Western blots of protein extracts from MCF - 7 transfected with YFP - p27 , YFP - p27T198A or YFP - p27T198D mutants following incubation with cycloheximide ( CHX ) for the indicated times . Uncropped images of the blots are shown in the Supplementary Information , Fig . S5 .

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(F) Western blots of protein extracts from MCF-7 transfected with YFP-p27, YFP-p27T198A or YFP-p27T198D mutants following incubation with cycloheximide (CHX) for the indicated times. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.


( a ) Crystal violet staining of MCF - 7 cell transfected with YFP ( C1 ) , YFP - p27 , YFP - p27T198A or YFP - p27T198D and selected with G418 for three weeks . Data are presented as mean ± s . d . ( n = 3 ) .

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(a) Crystal violet staining of MCF-7 cell transfected with YFP (C1), YFP-p27, YFP-p27T198A or YFP-p27T198D and selected with G418 for three weeks. Data are presented as mean ± s.d. (n = 3).


( b ) Flow cytometry analysis of MCF - 7 cells transfected with YFP ( C1 ) or YFP - p27 , YFP - p27T198A and YFP - p27T198D for 48 h . Data are presented as mean ± s . d . ( n = 3 ) .

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(b) Flow cytometry analysis of MCF-7 cells transfected with YFP (C1) or YFP-p27, YFP-p27T198A and YFP-p27T198D for 48 h. Data are presented as mean ± s.d. (n = 3).


( c ) GFP - LC3 U2OS cells 48 h post - transfection with RFP , RFP - p27 , RFP - p27T198A or RFP - p27T198D . Data represent results of three independent experiments , from which a total of ∼ 200 cells were counted ( s . d . , 10 ∼ 15 % ) . The scale bars represent 50 μm in c .

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(c) GFP-LC3 U2OS cells 48 h post-transfection with RFP, RFP-p27, RFP-p27T198A or RFP-p27T198D. Data represent results of three independent experiments, from which a total of ∼200 cells were counted (s.d., 10∼15%). The scale bars represent 50 μm in c.


( a , b ) MCF - 7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis ( a ) and cell - viability assays ( b ) .

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(a, b) MCF-7 cells were cultured for 24 h following siRNA with or without FBS before western blot analysis (a) and cell-viability assays (b).


( c ) MCF - 7 cells were transfected with vector alone ( C1 ) , YFP - p27 ( WT ) , or the siRNA - resistant constructs WT - R and T198A - R 24 h before RNA interference ( RNAi ) . PARP cleavage was determined by western blotting and densitometric scanning . Data represent three independent experiments ( mean ± s . d . ) .

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(c) MCF-7 cells were transfected with vector alone (C1), YFP-p27 (WT), or the siRNA-resistant constructs WT-R and T198A-R 24 h before RNA interference (RNAi). PARP cleavage was determined by western blotting and densitometric scanning. Data represent three independent experiments (mean ± s.d.).


( d ) Western blots of lysates of MCF - 7 cells transfected with siRNA before serum - starvation or treatment with LY294002 ( LY , 10 μM ) or rapamycin ( Rap , 100 nM ) for 48 h with or without methylpyruvate ( MP , 10 mM ) .

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(d) Western blots of lysates of MCF-7 cells transfected with siRNA before serum-starvation or treatment with LY294002 (LY, 10 μM) or rapamycin (Rap, 100 nM) for 48 h with or without methylpyruvate (MP, 10 mM).


( e ) MCF - 7 cells treated with or without LY294002 for 48 h following RNAi . p27 siRNA pool - 1 , siRNA1 , 2 and 3 ; arrows , membrane blebbing ; triangles , apoptotic bodies . The scale bar represents 50 μm .

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(e) MCF-7 cells treated with or without LY294002 for 48 h following RNAi. p27 siRNA pool-1, siRNA1, 2 and 3; arrows, membrane blebbing; triangles, apoptotic bodies. The scale bar represents 50 μm.


( f ) Western blots of protein extracts from MCF - 7 cultured with ( + ) or without ( − ) glucose for 24 h following RNAi . Fr , fragment ; percentage PARP cleavage = 100 × PARPFr / ( PARP + PARPFr ) .

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(f) Western blots of protein extracts from MCF-7 cultured with (+) or without (−) glucose for 24 h following RNAi. Fr, fragment; percentage PARP cleavage = 100 × PARPFr / (PARP + PARPFr).


( a ) Western blots of lysates from the indicated cell lines .

(a) Western blots of lysates from the indicated cell lines.


( b ) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors ( MDACC ) .

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(b) Pearson correlation analysis of p27 and LKB1 protein levels quantified by reverse phase protein array assays in 74 randomly selected primary breast tumors (MDACC).


( c ) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild - type LKB1 cultured with or without glucose for 24 h .

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(c) Western blots of protein lysates from Hela cells transfected with either pCDNA3 or wild-type LKB1 cultured with or without glucose for 24 h.


( d - f ) Western blots of protein extracts from MEFs treated with AICAR ( d and e ) ,

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(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e),


( d - f ) Western blots of protein extracts from MEFs treated with AICAR ( d and e )

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(d-f) Western blots of protein extracts from MEFs treated with AICAR (d and e)


( d - f ) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min ( f ) .

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(d-f) Western blots of protein extractsfrom 3T3L1 cells cultured with or without glucose for 30 min (f).


( g ) Western blot analysis of 3T3L1 cells 72 h post - transfection RNAi with or without glucose starvation for 1 h before analysis . Densitometric quantification of western blots is shown . Uncropped images of the blots are shown in the Supplementary Information , Fig . S5 .

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(g) Western blot analysis of 3T3L1 cells 72 h post-transfection RNAi with or without glucose starvation for 1 h before analysis. Densitometric quantification of western blots is shown. Uncropped images of the blots are shown in the Supplementary Information, Fig. S5.


( A ) FACS analysis of TUNEL positive p27 + / + and p27 − / − cells treated with AICAR for 20 h . Data represent mean ± s . d . of three independent experiments .

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(A) FACS analysis of TUNEL positive p27+/+ and p27−/− cells treated with AICAR for 20 h. Data represent mean ± s.d. of three independent experiments.


( B , C ) Electron microscopy of p27 + / + ( a , c and e ) and p27 − / − ( b , d and f ) MEFs treated with ( c and d ) , or without ( a and b ) AICAR ( 1 mM ) , or without glucose ( e and f ) for 14 h . Magnified images of the boxed areas were shown on the left ( a ′ , b ′ , c ′ , d ′ , e ′ and f ′ ) with arrowheads indicating autophagic vacuoles including autolysosomes ( black arrowheads ) . The scale bars represent 10 μm in a - f and 0 . 5 μm in a ′ - f ′ . N , nucleus . Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s . d . ( C ) .

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(B, C) Electron microscopy of p27+/+ (a, c and e) and p27−/− (b, d and f) MEFs treated with (c and d), or without (a and b) AICAR (1 mM), or without glucose (e and f) for 14 h. Magnified images of the boxed areas were shown on the left (a′, b′, c′, d′, e′ and f′) with arrowheads indicating autophagic vacuoles including autolysosomes (black arrowheads). The scale bars represent 10 μm in a-f and 0.5 μm in a′-f′. N, nucleus. Autophagic vacuoles were counted for three randomly selected cells from each electron microscopy section and data represent mean ± s.d. (C).


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 ) .

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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 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 ) .

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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 the dentate gyrus ( DG ) , cortex , CA1 , and CA3 regions of Arid1afl / + and cHet mice .

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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 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 ) .

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

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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 the Morris water maze assay . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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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 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 ) .

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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 / + 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 ) 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 .

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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 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 ) .

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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 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 .

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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 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 )

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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 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 ) .

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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 - 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 .

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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 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 .

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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 . 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 . )

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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.)


A In vitro differentiation to mature ookinete of wildtype ( WT ) , Δisp1 , Δisp3 , and Δisp1 / 3 parasites . Values are means ± SD ( n = 4 biological replicates ) ; two - tailed t test , * , P < 0 . 05 , * * , P < 0 . 01 , * * * , P < 0 . 001 , ns : not significant .

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A In vitro differentiation to mature ookinete of wildtype (WT), Δisp1, Δisp3, and Δisp1/3 parasites. Values are means ± SD (n=4 biological replicates); two-tailed t test, *, P<0.05, **, P<0.01, ***, P<0.001, ns: not significant.


Giemsa staining of zygotes / ookinetes from in vitro culture . Upper diagrams indicate morphological changes from zygote to ookinete . Black arrow indicates the apical . Scale bar = 5 μm .

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Giemsa staining of zygotes/ookinetes from in vitro culture. Upper diagrams indicate morphological changes from zygote to ookinete. Black arrow indicates the apical. Scale bar = 5 μm.


C Time - course analysis of ookinete differentiation from in vitro culture . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * * , P < 0 . 001 .

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C Time-course analysis of ookinete differentiation from in vitro culture. Values are means ± SEM (n=3 biological replicates), two-tailed t test, ***, P<0.001.


D In vivo ookinete differentiation in midgut of the infected mosquitos . Right panel indicates the parasite smear stained with Giemsa solution . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * , P < 0 . 01 . Scale bar = 5 μm .

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D In vivo ookinete differentiation in midgut of the infected mosquitos. Right panel indicates the parasite smear stained with Giemsa solution. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01. Scale bar = 5 μm.


E Number of oocysts in mosquito midgut 7 days post blood feeding . n is the number of mosquitoes in each group . The horizontal line shows the mean value of each group , Mann - Whitney test , * * * * , P < 0 . 0001 . Right panel shows mosquito midguts stained with 0 . 5 % mercurochrome . Scale bar = 50 μm . Three biological replicates performed .

E Number of oocysts in mosquito midgut 7 days post blood feeding. n is the number of mosquitoes in each group. The horizontal line shows the mean value of each group, Mann-Whitney test, ****, P<0.0001. Right panel shows mosquito midguts stained with 0.5% mercurochrome. Scale bar = 50 μm. Three biological replicates performed.


F Formation of salivary gland sporozoites in mosquitoes 14 days post blood feeding . In each group , at least twenty blood - fed mosquitoes were counted . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * , P < 0 . 01 .

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F Formation of salivary gland sporozoites in mosquitoes 14 days post blood feeding. In each group, at least twenty blood-fed mosquitoes were counted. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01.


G Western blot of ISP1 and ISP3 in zygote of the Δisp1 / 3 parasites episomally complemented with 3V5 - tagged isp1 or 6HA - tagged isp3 genes from either P . yoelii or P . falciparum . P28 as loading control . The DTS ( isp1 : : 3V5 ; isp3 : : 6HA ) is a doubly - tagged

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G Western blot of ISP1 and ISP3 in zygote of the Δisp1/3 parasites episomally complemented with 3V5-tagged isp1 or 6HA-tagged isp3 genes from either P. yoelii or P. falciparum. P28 as loading control. The DTS (isp1::3V5;isp3::6HA) is a doubly-tagged parasite


H IFA analysis of tagged ISP1 and ISP3 in zygotes of the complemented parasites . Nuclei are stained with Hoechst 33342 . Scale bar = 5 μm .

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H IFA analysis of tagged ISP1 and ISP3 in zygotes of the complemented parasites. Nuclei are stained with Hoechst 33342. Scale bar = 5 μm.


I In vitro ookinete differentiation of the complemented parasites . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * , P < 0 . 01 , * * * , P < 0 . 001 .

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I In vitro ookinete differentiation of the complemented parasites. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01, ***, P<0.001.


IFA of ISP1 and ISP3 expression from zygote to ookinete of the isp1 : : 6HA and isp3 : : 6HA parasites , respectively . Scale bar = 5 μm .

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IFA of ISP1 and ISP3 expression from zygote to ookinete of the isp1::6HA and isp3::6HA parasites, respectively. Scale bar = 5 μm.


B Co - localization of ISP1 and ISP3 from zygote to ookinete in the doubly - tagged strain ( DTS , isp1 : : 3V5 ; isp3 : : 6HA ) . Scale bar = 5 μm .

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B Co-localization of ISP1 and ISP3 from zygote to ookinete in the doubly-tagged strain (DTS, isp1::3V5;isp3::6HA). Scale bar = 5 μm.


C Western blot of ISP1 and ISP3 expression in zygotes of isp1 : : 6HA and isp3 : : 6HA parasites . The numbers are the relative intensities of band in blot . Right panel : the quantification of band intensity . Values are means ± SEM ( n = 4 biological replicates ) , two - tailed t test , * * * * , P < 0 . 0001 .

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C Western blot of ISP1 and ISP3 expression in zygotes of isp1::6HA and isp3::6HA parasites. The numbers are the relative intensities of band in blot. Right panel: the quantification of band intensity. Values are means ± SEM (n=4 biological replicates), two-tailed t test, ****, P<0.0001.


D Western blot of ISP1 and ISP3 expression in zygotes of DTS , DTS / Δisp1 , and DTS / Δisp3 parasites . The numbers are the relative intensities of band in blot . Three replicates performed .

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D Western blot of ISP1 and ISP3 expression in zygotes of DTS, DTS/Δisp1, and DTS/Δisp3 parasites. The numbers are the relative intensities of band in blot. Three replicates performed.


E RT - qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT , Δisp1 , Δisp3 , and Δisp1 / 3 parasites . The expression is normalized to the 18s rRNA . Values are means ± SEM ( n = 4 biological replicates ) .

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E RT-qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT, Δisp1, Δisp3, and Δisp1/3 parasites. The expression is normalized to the 18s rRNA. Values are means ± SEM (n=4 biological replicates).


F IFA analysis of ISP1 and ISP3 expression dynamic of DTS / Δisp1 and DTS / Δisp3 parasites . Scale bar = 5 μm .

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F IFA analysis of ISP1 and ISP3 expression dynamic of DTS/Δisp1 and DTS/Δisp3 parasites. Scale bar = 5 μm.


B Fluorescent microscopy of ISP and its N - terminal 20 residues ( N20 ) fused with a BFP : : 3V5 peptide episomally expressed in zygotes . Scale bar = 5 μm .

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B Fluorescent microscopy of ISP and its N-terminal 20 residues (N20) fused with a BFP::3V5 peptide episomally expressed in zygotes. Scale bar = 5 μm.


C Solubility assay detecting membrane association of ISP1 _ FL and ISP1 _ N20 using different detergents . Cytosolic soluble proteins are in hypotonic buffer ( Hypo ) , peripheral membrane proteins in carbonate buffer ( Carb ) ， integral membrane proteins in Triton X - 100 buffer ( Trx ) , and insoluble proteins in pellet ( P ) . BFP : : 3V5 is in the soluble fraction , while ISP1 _ FL and ISP1 _ N20 are in the membrane - associated fractions . ER protein BiP is a loading control .

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

C Solubility assay detecting membrane association of ISP1_FL and ISP1_N20 using different detergents. Cytosolic soluble proteins are in hypotonic buffer (Hypo), peripheral membrane proteins in carbonate buffer (Carb)，integral membrane proteins in Triton X-100 buffer (Trx), and insoluble proteins in pellet (P). BFP::3V5 is in the soluble fraction, while ISP1_FL and ISP1_N20 are in the membrane-associated fractions. ER protein BiP is a loading control.


D Acyl - RAC method detecting palmitoylation of ISP1 and ISP3 in zygotes of the DTS strain . Proteins from F ( flow - through ) , W ( wash ) , and E ( elution ) are analyzed . P28 as a loading control .

O O O O O O O O B-MEASURED_VAR 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

D Acyl-RAC method detecting palmitoylation of ISP1 and ISP3 in zygotes of the DTS strain. Proteins from F (flow-through), W (wash), and E (elution) are analyzed. P28 as a loading control.


E Substitutions of N - terminal cysteine or glycine residues to alanine ( G2A or C7A / C8A ) ablates palmitoylation of ISP1 . ISP1 was fused with BFP : : 3V5 and episomally expressed in zygotes . Right panel shows quantification of palmitoylated protein band from two independent repeats , values are means ± SD .

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-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 Substitutions of N-terminal cysteine or glycine residues to alanine (G2A or C7A/C8A) ablates palmitoylation of ISP1. ISP1 was fused with BFP::3V5 and episomally expressed in zygotes. Right panel shows quantification of palmitoylated protein band from two independent repeats, values are means ± SD.


F G2A and C7AC8A mutations cause cytoplasmic distribution of ISP1 during zygote to ookinete differentiation . Scale bar = 5 μm .

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F G2A and C7AC8A mutations cause cytoplasmic distribution of ISP1 during zygote to ookinete differentiation. Scale bar = 5 μm.


G Membrane association of ISP1 with either G2A or C7AC8A substitutions .

G Membrane association of ISP1 with either G2A or C7AC8A substitutions.


H Expression and palmitoylation of ISP1 in ∆ isp1 / 3 parasites complemented with the 3V5 tagged WT ISP1 or C7AC8A mutant .

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

H Expression and palmitoylation of ISP1 in ∆isp1/3 parasites complemented with the 3V5 tagged WT ISP1 or C7AC8A mutant.


I IFA of ISP1 at zygote of the complemented parasites . Scale bar = 5 μm .

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

I IFA of ISP1 at zygote of the complemented parasites. Scale bar = 5 μm.


In vitro differentiation to mature ookinete of the complemented parasites . Values are means ± SEM ( n = 4 replicates ) , two - tailed t test , * * * , P < 0 . 001 , ns : not significant .

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

In vitro differentiation to mature ookinete of the complemented parasites. Values are means ± SEM (n=4 replicates), two-tailed t test, ***, P<0.001, ns: not significant.


A Two - colored IFA of ISP1 / DHHC2 and ISP3 / DHHC2 at zygotes of the triple tagged strain isp1 : : 3V5 / isp3 : : 6HA / dhhc2 : : 4Myc ( TTS ) . Right graph shows overlay of fluorescence peaks along cell periphery . Scale bar = 5 μm .

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A Two-colored IFA of ISP1/DHHC2 and ISP3/DHHC2 at zygotes of the triple tagged strain isp1::3V5/isp3::6HA/dhhc2::4Myc (TTS). Right graph shows overlay of fluorescence peaks along cell periphery. Scale bar = 5 μm.


B Western blot of DHHC2 , ISP1 , and P28 ( plasma membrane protein ) of the TTS zygotes treated with PBS , trypsin ( Try ) , or heat - inactivated ( HI ) trypsin . Upper panel shows the PPM and IMC structures .

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

B Western blot of DHHC2, ISP1, and P28 (plasma membrane protein) of the TTS zygotes treated with PBS, trypsin (Try), or heat-inactivated (HI) trypsin. Upper panel shows the PPM and IMC structures.


C Knockdown of dhhc2 expression . Upper panel shows the promoter swap strategy applied in TTS strain , generating the dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter . Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes . Three replicates performed .

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

C Knockdown of dhhc2 expression. Upper panel shows the promoter swap strategy applied in TTS strain, generating the dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter. Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes. Three replicates performed.


D Two - colored IFA of DHHC2 and P28 proteins in the TTS and dhhc2kd zygotes . Scale bar = 5 μm . Bottom panel , quantifications of DHHC2 fluorescence . Values are means ± SD ( n : the number of cells analyzed ) . Mann - Whitney test , * * * * , P < 0 . 0001 .

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

D Two-colored IFA of DHHC2 and P28 proteins in the TTS and dhhc2kd zygotes. Scale bar = 5 μm. Bottom panel, quantifications of DHHC2 fluorescence. Values are means ± SD (n: the number of cells analyzed). Mann-Whitney test, ****, P<0.0001.


E Western blot of DHHC2 , ISP1 and ISP3 in the TTS and dhhc2kd zygotes . Two replicates performed .

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

E Western blot of DHHC2, ISP1 and ISP3 in the TTS and dhhc2kd zygotes. Two replicates performed.


F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl - RAC method . NH : NH2OH . Right panel indicates quantification of protein band intensity from three replicates , values are means ± SD .

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

F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl-RAC method. NH: NH2OH. Right panel indicates quantification of protein band intensity from three replicates, values are means ± SD.


G IFA of ISP1 and ISP3 in zygotes of TTS and dhhc2kd . Scale bar = 5 μm .

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

G IFA of ISP1 and ISP3 in zygotes of TTS and dhhc2kd. Scale bar = 5 μm.


H Giemsa staining of zygote / ookinete from in vitro cultured TTS and dhhc2kd parasites . Scale bar = 5 μm .

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

H Giemsa staining of zygote/ookinete from in vitro cultured TTS and dhhc2kd parasites. Scale bar = 5 μm.


I Time - course analysis of in vitro ookinete differentiation . Values are means ± SD from three independent tests .

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

I Time-course analysis of in vitro ookinete differentiation. Values are means ± SD from three independent tests.


J IFA of DHHC2 expression in the dhhc2 : : 6HA and dhhc2 : : 6HA ; ∆ isp1 / 3 zygotes . Scale bar = 5 μm .

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 O O O O O

J IFA of DHHC2 expression in the dhhc2::6HA and dhhc2::6HA;∆isp1/3 zygotes. Scale bar = 5 μm.


K Western blot of DHHC2 in zygotes of dhhc2 : : 6HA and dhhc2 : : 6HA ; ∆ isp1 / 3 .

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

K Western blot of DHHC2 in zygotes of dhhc2::6HA and dhhc2::6HA;∆isp1/3.


L IFA of DHHC2 and ISP1 in zygotes of dhhc2kd parasites complemented with 4Myc tagged WT PyDHHC2 of P . yoelii , PfDHHC2 of P . falciparum , or PyDHHC2 with PAT catalytic deficient mutation C128A . Scale bar = 5 μm .

O O O B-MEASURED_VAR 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 O O O O O

L IFA of DHHC2 and ISP1 in zygotes of dhhc2kd parasites complemented with 4Myc tagged WT PyDHHC2 of P. yoelii, PfDHHC2 of P. falciparum, or PyDHHC2 with PAT catalytic deficient mutation C128A. Scale bar = 5 μm.


M Western blot of DHHC2 in the complemented dhhc2kd parasites as indicated .

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

M Western blot of DHHC2 in the complemented dhhc2kd parasites as indicated.


N In vitro differentiation to mature ookinetes of the complemented parasites . Values are means ± SD from three independent tests . two - tailed t test , ns : not significant .

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

N In vitro differentiation to mature ookinetes of the complemented parasites. Values are means ±SD from three independent tests. two-tailed t test, ns: not significant.


Western blot of episomally expressed DHHC2 : : 6HA or variants containing truncated segment ( C1 : 210 - 232 , C2 : 233 - 262 , C3 : 263 - 284 residues ) in WT zygotes .

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

Western blot of episomally expressed DHHC2::6HA or variants containing truncated segment (C1:210-232, C2:233-262, C3:263-284 residues) in WT zygotes.


C IFA of episomally expressed DHHC2 : : 6HA and three truncation mutants in zygotes and retorts . Scale bar = 5 μm .

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

C IFA of episomally expressed DHHC2::6HA and three truncation mutants in zygotes and retorts. Scale bar = 5 μm.


D Acyl - RAC method detecting palmitoylation of endogenous DHHC2 in the dhhc2 : : 6HA zygotes .

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

D Acyl-RAC method detecting palmitoylation of endogenous DHHC2 in the dhhc2::6HA zygotes.


E Palmitoylation of DHHC2 : : 6HA and C4A mutant episomally expressed in zygotes . C4A : substitutions of C - terminal four cysteine to alanine . Right panel indicates the quantification of protein band intensity from two replicates , values are means ± SD .

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

E Palmitoylation of DHHC2::6HA and C4A mutant episomally expressed in zygotes. C4A: substitutions of C-terminal four cysteine to alanine. Right panel indicates the quantification of protein band intensity from two replicates, values are means ± SD.


F Cytoplasmic distribution of C4A mutant during zygote to ookinete differentiation . Scale bar = 5 μm .

F Cytoplasmic distribution of C4A mutant during zygote to ookinete differentiation. Scale bar = 5 μm.


G IFA of DHHC2 at the dhhc2 : : 6HA zygotes treated with 100 μΜ 2 - BP . Scale bar = 5 μm .

O O O B-MEASURED_VAR 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

G IFA of DHHC2 at the dhhc2::6HA zygotes treated with 100 μΜ 2-BP. Scale bar = 5 μm.


H In vitro ookinete differentiation of the dhhc2kd parasites complemented with either WT DHHC2 or C4A mutant . Values are means ± SD from three independent tests .

O O O O O O 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 O

H In vitro ookinete differentiation of the dhhc2kd parasites complemented with either WT DHHC2 or C4A mutant. Values are means ± SD from three independent tests.


I Palmitoylation of the episomally expressed DHHC2 : : 6HA with mutation in respective individual cysteine ( C255A , C258A , C260A , and C262A ) . Representative of three independent repeats .

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

I Palmitoylation of the episomally expressed DHHC2::6HA with mutation in respective individual cysteine (C255A, C258A, C260A, and C262A). Representative of three independent repeats.


J Palmitoylation of HA - tagged and human codon optimized DHHC2 ectopically expressed in human HEK293T cells . Both C4A and C128A mutations ablate DHHC2 palmitoylation .

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 B-CONTROLLED_VAR O O

J Palmitoylation of HA-tagged and human codon optimized DHHC2 ectopically expressed in human HEK293T cells. Both C4A and C128A mutations ablate DHHC2 palmitoylation.


K DHHC2 _ C128A - HA mutant protein could be palmitoylated by the co - transfected DHHC2WT - Myc , but not DHHC2 _ C128A - Myc in HEK293T cells . Representative of two independent repeats .

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

K DHHC2_C128A-HA mutant protein could be palmitoylated by the co-transfected DHHC2WT-Myc, but not DHHC2_C128A-Myc in HEK293T cells. Representative of two independent repeats.


L Co - immunoprecipitation of the DHHC2 _ WT - HA and DHHC2 _ WT - Myc co - expressed in HEK293T cells .

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

L Co-immunoprecipitation of the DHHC2_WT-HA and DHHC2_WT-Myc co-expressed in HEK293T cells.


M C - terminus 75 residues peptide ( Cter75 ) of DHHC2 could be palmitoylated as an independent substrate by co - transfected DHHC2WT - HA in HEK293T cells . Cter75 is fused with a BFP : : Myc for easy detection via western blot . Cter75 * peptide contains C4A mutation .

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 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

M C-terminus 75 residues peptide (Cter75) of DHHC2 could be palmitoylated as an independent substrate by co-transfected DHHC2WT-HA in HEK293T cells. Cter75 is fused with a BFP::Myc for easy detection via western blot. Cter75* peptide contains C4A mutation.


N Palmitoylation of tagged DHHC2 ectopically expressed in bacteria E . coli .

N Palmitoylation of tagged DHHC2 ectopically expressed in bacteria E.coli.


O Palmitoylation of DHHC2 ectopically expressed in bacteria treated with 2 - BP .

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

O Palmitoylation of DHHC2 ectopically expressed in bacteria treated with 2-BP.


B Endogenous DHHC2 is palmitoylated in the dhhc2 : : 6HA gametocytes .

B Endogenous DHHC2 is palmitoylated in the dhhc2::6HA gametocytes.


C Episomally expressed DHHC2 driven by promoter of ccp2 gene is palmitoylated in female gametocytes , female gametes , and zygotes of WT parasites . Left panel shows the expression of HA - tagged DHHC2 in female gametocyte by co - staining with antibodies of HA and α - Tubulin II ( male gametocyte specific ) . Scale bar = 5 μm .

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

C Episomally expressed DHHC2 driven by promoter of ccp2 gene is palmitoylated in female gametocytes, female gametes, and zygotes of WT parasites. Left panel shows the expression of HA-tagged DHHC2 in female gametocyte by co-staining with antibodies of HA and α-Tubulin II (male gametocyte specific). Scale bar = 5 μm.


D IFA of DHHC2 and P28 in female gametocytes , female gametes , and zygotes of the dhhc2 : : 6HA parasite . Gametocytes were stimulated with XA and 22 ° C in vitro . P28 is expressed specifically in female gametes and zygotes . Scale bar = 5 μm .

O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR 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

D IFA of DHHC2 and P28 in female gametocytes, female gametes, and zygotes of the dhhc2::6HA parasite. Gametocytes were stimulated with XA and 22°C in vitro. P28 is expressed specifically in female gametes and zygotes. Scale bar = 5 μm.


E Quantification of parasites with DHHC2 polarization in P28 positive cells during gametocyte to zygote differentiation x / y : the number of cells with DHHC2 polarization and the number of cells analyzed . Values are means ± SD from three independent tests .

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

E Quantification of parasites with DHHC2 polarization in P28 positive cells during gametocyte to zygote differentiation x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ±SD from three independent tests.


F No polarization of DHHC2 in the dhhc2 : : 6HA gametocytes with only either XA or 22 ° C stimulation . Three replicates performed , values are means ± SD .

O 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

F No polarization of DHHC2 in the dhhc2::6HA gametocytes with only either XA or 22°C stimulation. Three replicates performed, values are means ± SD.


G No change in DHHC2 expression in zygotes of the dhhc2 : : 6HA parasite - derived mutants with individual gene disruption of cdpk4 , hap2 , and dozi .

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

G No change in DHHC2 expression in zygotes of the dhhc2::6HA parasite-derived mutants with individual gene disruption of cdpk4, hap2, and dozi.


H Quantification of parasite with DHHC2 polarization in P28 positive cells . Parasites are indicated in ( G ) or from a genetic cross ( dhhc2 : : 6HA / cdpk4 and WT ) . x / y : the number of cells with DHHC2 polarization and the number of cells analyzed . Values are means ± SD from three tests , two - tailed t test , * * * , P < 0 . 001 , * * * * , P < 0 . 0001 , ns , not significant .

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

H Quantification of parasite with DHHC2 polarization in P28 positive cells. Parasites are indicated in (G) or from a genetic cross (dhhc2::6HA/cdpk4 and WT). x/y: the number of cells with DHHC2 polarization and the number of cells analyzed. Values are means ± SD from three tests, two-tailed t test, ***, P<0.001, ****, P<0.0001, ns, not significant.


I Expression and localization of DHHC2 and GAP45 during female gamete to ookinete development of dhhc2 : : 6HA parasite . GAP45 is an IMC marker and is only detected in zygote after fertilization , but not gametes . Scale bar = 5 μm .

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

I Expression and localization of DHHC2 and GAP45 during female gamete to ookinete development of dhhc2::6HA parasite. GAP45 is an IMC marker and is only detected in zygote after fertilization, but not gametes. Scale bar = 5 μm.


A Transmission electron microscopy ( TEM ) cross sections of WT , dhhc2kd and ∆ isp1 / 3 parasites showing the arrangement of SPM underneath IMC . Approximately 60 recognizable hollow microtubules ( green ) are associated with IMC ( red ) , distributing evenly around circumference with an interval of 100 - 120 nm in WT ookinetes . Microtubules lost association with IMC in the ookinetes of dhhc2kd and ∆ isp1 / 3 parasites . Left : one representative cell in each parasite is shown ; Right : two areas are magnified to show the details . Lower panel is the quantification of distance between adjacent microtubules associated with IMC . Values are mean ± SD for n = 204 ( WT ) , 189 ( dhhc2kd ) , and 211 ( ∆ isp1 / 3 ) measurements in 20 cells each group from two independent experiments ; Kolmogorov - Smirnov test , * * * * , P < 0 . 0001 .

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

A Transmission electron microscopy (TEM) cross sections of WT, dhhc2kd and ∆isp1/3 parasites showing the arrangement of SPM underneath IMC. Approximately 60 recognizable hollow microtubules (green) are associated with IMC (red), distributing evenly around circumference with an interval of 100-120 nm in WT ookinetes. Microtubules lost association with IMC in the ookinetes of dhhc2kd and ∆isp1/3 parasites. Left: one representative cell in each parasite is shown; Right: two areas are magnified to show the details. Lower panel is the quantification of distance between adjacent microtubules associated with IMC. Values are mean ± SD for n=204 (WT), 189 (dhhc2kd), and 211 (∆isp1/3) measurements in 20 cells each group from two independent experiments; Kolmogorov-Smirnov test, ****, P<0.0001.


B Apical polar ring and the emanating SPM in WT , dhhc2kd and ∆ isp1 / 3 parasites examined by negative staining and TEM . A dome - like SPM structure radiating from the apical polar ring was observed in early ( 3 hours ) or mature ( 12 hours ) WT ookinetes , but impaired in mutant parasites . n is the number of cells analyzed from three independent experiments . More images in the Appendix Figure S5 . Scale bar = 1μm .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Apical polar ring and the emanating SPM in WT, dhhc2kd and ∆isp1/3 parasites examined by negative staining and TEM. A dome-like SPM structure radiating from the apical polar ring was observed in early (3 hours) or mature (12 hours) WT ookinetes, but impaired in mutant parasites. n is the number of cells analyzed from three independent experiments. More images in the Appendix Figure S5. Scale bar = 1μm.


C SiR - tubulin staining show the SPM structures of WT , dhhc2kd and ∆ isp1 / 3 parasites . Arrowheads point to cell apical . Scale bar = 5 μm .

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C SiR-tubulin staining show the SPM structures of WT, dhhc2kd and ∆isp1/3 parasites. Arrowheads point to cell apical. Scale bar = 5 μm.


A GST - ISP1 pulls down endogenous β - Tubulin from cell lysate of WT ookinetes . Lower panel indicates the Coomassie blue stain of recombinant GST and GST - ISP1 .

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 B-MEASURED_VAR O

A GST-ISP1 pulls down endogenous β-Tubulin from cell lysate of WT ookinetes. Lower panel indicates the Coomassie blue stain of recombinant GST and GST-ISP1.


B Episomally expressed ISP1 : : HA immunoprecipitated with β - Tubulin in WT ookinetes . Episomal vector as control .

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

B Episomally expressed ISP1::HA immunoprecipitated with β-Tubulin in WT ookinetes. Episomal vector as control.


C Endogenous ISP1 : : 3V5 immunoprecipitated with β - Tubulin in the TTS ookinetes .

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C Endogenous ISP1::3V5 immunoprecipitated with β-Tubulin in the TTS ookinetes.


D Co - localization of ISP1 : : HA and β - Tubulin at ookinete periphery in IFA . Scale bar = 5 μm .

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D Co-localization of ISP1::HA and β-Tubulin at ookinete periphery in IFA. Scale bar = 5 μm.


E Proximity ligation assay ( PLA ) detecting interaction of ISP1 - HA and β - Tubulin at ookinete . Episomal vector as control . Scale bar = 5 μm .

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E Proximity ligation assay (PLA) detecting interaction of ISP1-HA and β-Tubulin at ookinete. Episomal vector as control. Scale bar = 5 μm.


F Solubility assay detecting membrane association of α - and β - Tubulins in WT , dhhc2kd , and ∆ isp1 / 3 parasites . GAPDH is a cytosol soluble protein , P28 is a plasma membrane integral protein .

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F Solubility assay detecting membrane association of α- and β-Tubulins in WT, dhhc2kd, and ∆isp1/3 parasites. GAPDH is a cytosol soluble protein, P28 is a plasma membrane integral protein.


G Quantification of α - and β - Tubulins membrane association in F . The membrane association index is defined as the ratio of Carb fraction to Hypo fraction in F , and normalized to that of WT . Values are means ± SEM from three independent tests .

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G Quantification of α- and β-Tubulins membrane association in F. The membrane association index is defined as the ratio of Carb fraction to Hypo fraction in F, and normalized to that of WT. Values are means ± SEM from three independent tests.


H Linear correlation of membrane association index of α - Tubulin and β - Tubulin with the conversion rate to mature ookinete among WT , dhhc2kd and ∆ isp1 / 3 parasites . Horizontal and vertical values are means ± SD .

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H Linear correlation of membrane association index of α-Tubulin and β-Tubulin with the conversion rate to mature ookinete among WT, dhhc2kd and ∆isp1/3 parasites. Horizontal and vertical values are means ± SD.


I Membrane association of α - and β - Tubulins in WT ookinete culture after DMSO , 2 - BP , or colchicine treatment .

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I Membrane association of α- and β-Tubulins in WT ookinete culture after DMSO, 2-BP, or colchicine treatment.


J Quantification of α - and β - Tubulins membrane association Values are means ± SEM from three independent repeats .

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J Quantification of α- and β-Tubulins membrane association Values are means ± SEM from three independent repeats.


C . cGAMP - VLPs induce an IFN - I response in target cells . HEK293 cells were infected with decreasing amounts of cGAMP - VLPs and Empty - VLPs ( 1 / 5 serial dilutions starting at 2μL of VLP stocks per well ) and the infection was monitored 24 hours later by quantifying GFP + cells by flow cytometry . Supernatants from the same infected cells were then transferred to a reporter cell line expressing firefly luciferase under a promoter induced by IFN - I ( ISRE ) . Luciferase activity measured 24 hours later indicated the presence of IFN - I in the supernatants .

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C. cGAMP-VLPs induce an IFN-I response in target cells. HEK293 cells were infected with decreasing amounts of cGAMP-VLPs and Empty-VLPs (1/5 serial dilutions starting at 2μL of VLP stocks per well) and the infection was monitored 24 hours later by quantifying GFP+ cells by flow cytometry. Supernatants from the same infected cells were then transferred to a reporter cell line expressing firefly luciferase under a promoter induced by IFN-I (ISRE). Luciferase activity measured 24 hours later indicated the presence of IFN-I in the supernatants.


Immunisation with cGAMP - VLPs facilitates induction of HIV - 1 Gag - specific polyfunctional CD8 T cell responses . Alternatively , cells were analysed by ICS six hours after stimulation with peptide . CD8 T cells were gated as live , CD90 . 2 + , CD8 + . CD8 T cells expressing CD107a , IFNγ , TNFα or IL2 were analysed as shown in ( E ) .

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Immunisation with cGAMP-VLPs facilitates induction of HIV-1 Gag-specific polyfunctional CD8 T cell responses. Alternatively, cells were analysed by ICS six hours after stimulation with peptide. CD8 T cells were gated as live, CD90.2+, CD8+. CD8 T cells expressing CD107a, IFNγ, TNFα or IL2 were analysed as shown in (E).


A , B . cGAMP loading enhances IgG responses specific to VLP proteins , including VSV - G . ELISA plates were coated with lysate from cGAMP - VLPs ( A ) or recombinant VSV - G protein ( B ) . Antibodies of different isotypes specific for these proteins were measured in sera from immunised mice by ELISA . The optical density at increasing serum dilutions is shown in the first three graphs from the left and the EC50 is on the right .

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A, B. cGAMP loading enhances IgG responses specific to VLP proteins, including VSV-G. ELISA plates were coated with lysate from cGAMP-VLPs (A) or recombinant VSV-G protein (B). Antibodies of different isotypes specific for these proteins were measured in sera from immunised mice by ELISA. The optical density at increasing serum dilutions is shown in the first three graphs from the left and the EC50 is on the right.


C . Enhanced antibody production following immunisation with cGAMP - VLPs relies on STING signalling . WT or Sting - / - mice were immunised with PBS , cGAMP - VLPs , Empty - VLPs or Empty - VLPs + poly ( I : C ) . IgG2b antibodies recognising VLP proteins were assessed by ELISA . The optical density at increasing serum dilutions is shown in the first two graphs from the left and the EC50 is on the right .

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C. Enhanced antibody production following immunisation with cGAMP-VLPs relies on STING signalling. WT or Sting-/- mice were immunised with PBS, cGAMP-VLPs, Empty-VLPs or Empty-VLPs + poly(I:C). IgG2b antibodies recognising VLP proteins were assessed by ELISA. The optical density at increasing serum dilutions is shown in the first two graphs from the left and the EC50 is on the right.


Immunisation with cGAMP - VLPs enhances accumulation of Tfh cells in the draining lymph node . T follicular ( Tf ) cells were identified by flow cytometry as CD4 + CD44 + CXCR5hiPD1hi cells and were further subdivided into Tfr cells ( FoxP3 + ) and Tfh cells ( FoxP3 - ) . the percentages of Tf , Tfh and Tfr cells within CD4 + cells are shown in ( B ) .

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Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B).


Immunisation with VLPs induces germinal centre formation . Germinal centre B cells were identified by flow cytometry as B220 + IgD - CD95 + GL7 + cells . the percentage of germinal centre B cells amongst B220 + cells is shown in ( E ) .

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Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. the percentage of germinal centre B cells amongst B220+ cells is shown in (E).


C , D . Low doses of cGAMP - HA - VLPs confer protection following IAV challenge . One month after immunisation with the indicated doses of VLPs , animals were infected with 104 TCID50 of IAV PR8 virus . Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight ( C , upper graph shows mean and lower graphs show individual mice for each condition ) . Animals approaching the humane end - point of 20 % weight loss were culled and survival to end - point curves are shown in ( D ) .

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C, D. Low doses of cGAMP-HA-VLPs confer protection following IAV challenge. One month after immunisation with the indicated doses of VLPs, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (C, upper graph shows mean and lower graphs show individual mice for each condition). Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (D).


B . cGAMP - HA - VLPs and Empty - HA - VLPs + AddaVax protect against IAV challenge . One month after immunisation , animals were infected with 104 TCID50 of IAV PR8 virus . Animals approaching the humane end - point of 20 % weight loss were culled and survival to end - point curves are shown in ( B ) .

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B. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (B).


A . cGAMP - S - VLPs and Empty - S - VLPs incorporate SARS - CoV - 2 S . Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS - CoV - 2 S , Gag - GFP and actin using the indicated antibodies .

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A. cGAMP-S-VLPs and Empty-S-VLPs incorporate SARS-CoV-2 S. Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS-CoV-2 S, Gag-GFP and actin using the indicated antibodies.


B , C . Immunisation with cGAMP - S - VLPs augments anti - Spike and anti - RBD antibody titres . Mice were immunised via the intra - muscular route with 5x105 IU of cGAMP - S - VLPs , Empty - S - VLPs , or PBS as a control . Three weeks after immunisation , sera were collected , heat - inactivated , and titres of antibodies capable of binding to SARS - CoV - 2 S ( B ) or its RBD ( C ) were determined by ELISA . The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal ( blank + 10SD ) . The dotted line shows the limit of detection ( LOD ) .

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B, C. Immunisation with cGAMP-S-VLPs augments anti-Spike and anti-RBD antibody titres. Mice were immunised via the intra-muscular route with 5x105 IU of cGAMP-S-VLPs, Empty-S-VLPs, or PBS as a control. Three weeks after immunisation, sera were collected, heat-inactivated, and titres of antibodies capable of binding to SARS-CoV-2 S (B) or its RBD (C) were determined by ELISA. The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10SD). The dotted line shows the limit of detection (LOD).


D . Immunisation with cGAMP - S - VLPs induces neutralising antibodies . Using serum samples from ( B ) , antibody titres capable of neutralising SARS - CoV - 2 were determined by microneutralisation ( MN ) assay . Calculated IC50 doses from multiple serum dilutions are shown . The dotted line shows the LOD .

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D. Immunisation with cGAMP-S-VLPs induces neutralising antibodies. Using serum samples from (B), antibody titres capable of neutralising SARS-CoV-2 were determined by microneutralisation (MN) assay. Calculated IC50 doses from multiple serum dilutions are shown. The dotted line shows the LOD.


C . Tissue distribution of the Ldb2 protein . Representative western - blot image is shown . Yellow asterisks indicate uncharacterized bands .

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C. Tissue distribution of the Ldb2 protein. Representative western-blot image is shown. Yellow asterisks indicate uncharacterized bands.


D . Expression of Ldb2 in the cerebral cortex . DAPI - stained nuclei visualized ( right ) in the cerebral cortex .

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D. Expression of Ldb2 in the cerebral cortex. DAPI-stained nuclei visualized (right) in the cerebral cortex.


E . Expression of Ldb2 in the amygdala . LA ; lateral amygdala , BLA ; basolateral amygdala , Ce ; central nucleus of amygdala .

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E. Expression of Ldb2 in the amygdala. LA; lateral amygdala, BLA; basolateral amygdala, Ce; central nucleus of amygdala.


F - G . Expression of Ldb2 in neurons . The merged images obtained with Ldb2 ( stained in green ) and NeuN ( stained in red ) antibodies are shown for cerebral cortex layers I - VI and the amygdala ( F ) . most of Ldb2 - positive cells are NeuN - positive . G . NeuN - positive cells / Ldb2 - positive cells ( left ) and Ldb2 - positive cells / NeuN - positive cells were counted in cerebral cortex layers I - VI .

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F-G. Expression of Ldb2 in neurons. The merged images obtained with Ldb2 (stained in green) and NeuN (stained in red) antibodies are shown for cerebral cortex layers I-VI and the amygdala (F). most of Ldb2-positive cells are NeuN-positive. G. NeuN-positive cells/Ldb2-positive cells (left) and Ldb2-positive cells/NeuN-positive cells were counted in cerebral cortex layers I-VI.


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Home cage - activity test ( A ) . Voluntary locomotor activities in the home cages were monitored for a week . Data of dark ( left ) and light ( right ) phases are indicated .

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Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Home cage-activity test (A). Voluntary locomotor activities in the home cages were monitored for a week. Data of dark (left) and light (right) phases are indicated.


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Open - field test ( B )

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Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Open-field test (B)


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Acoustic startle response ( C )

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Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Acoustic startle response (C)


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Prepulse - inhibition test ( D )

Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Prepulse-inhibition test (D)


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in , Morris water - maze test ( E ) where data in hidden test ( escape latency and moving speed , path length and no movement time ) and probe test ( path length ) are

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Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in , Morris water-maze test (E) where data in hidden test (escape latency and moving speed, path length and no movement time) and probe test (path length) are shown


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Y - maze test ( F ) .

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Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Y-maze test (F).


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Fear - conditioning test ( G ) . Results of contextual ( left ) and cued ( right ) tests are shown .

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Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Fear-conditioning test (G). Results of contextual (left) and cued (right) tests are shown.


Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Hot - plate test ( H ) .

Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Hot-plate test (H).


Locomotor activity was assessed in the same experimental set as in the open - field test . After acclimation for 120 min , mice ( n = 10 - 12 / group ) were injected ( arrows ) with methylphenidate ( MPD : 3 mg / kg , A ) and their locomotor activity was monitored . In the right panels , cumulative values for pre - and post - injection times are also shown .

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Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with methylphenidate (MPD: 3 mg/kg, A) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.


Locomotor activity was assessed in the same experimental set as in the open - field test . After acclimation for 120 min , mice ( n = 10 - 12 / group ) were injected ( arrows ) with MK - 801 ( 0 . 2 mg / kg , B ) and their locomotor activity was monitored . In the right panels , cumulative values for pre - and post - injection times are also shown .

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Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with MK-801 (0.2 mg/kg, B) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.


C . Popping behavior induced by the injection of MK - 801 ( 0 . 5 mg / kg ) was manually assessed in WT ( n = 4 ) and KO ( n = 6 ) animals . Typically , each episode is constituted by blocks of 5 - 15 successive jumping behaviors . Episode numbers in 20 min ( left ) and total duration ( right ) are shown in a box - whisker plot .

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C. Popping behavior induced by the injection of MK-801 (0.5 mg/kg) was manually assessed in WT (n = 4) and KO (n = 6) animals. Typically, each episode is constituted by blocks of 5-15 successive jumping behaviors. Episode numbers in 20 min (left) and total duration (right) are shown in a box-whisker plot.


A . Fear - conditioning test . Freezing ( % ) was monitored in the cued ( top ) and contextual ( bottom ) tests before ( pre ) and after ( post ) administration of clozapine ( left ) or haloperidol ( right ) . Data are presented as means ± S . E . M . ( n = 7 - 9 / group ) . * , p < 0 . 05 ; * * * , p < 0 . 001 ( Tukey test ) ; # , p < 0 . 05 ( t - test , KO / saline vs . KO / clozapine ) .

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A. Fear-conditioning test. Freezing (%) was monitored in the cued (top) and contextual (bottom) tests before (pre) and after (post) administration of clozapine (left) or haloperidol (right). Data are presented as means ± S.E.M. (n = 7-9/group). *, p < 0.05; ***, p < 0.001 (Tukey test); #, p < 0.05 (t-test, KO/saline vs. KO/clozapine).