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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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


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

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


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

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

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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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

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 ) .

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

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 ) .

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

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).


B . Home cage - activity test . Spontaneous locomotor activity of mice chronically fed chow with or without 0 . 24 % ( w / w ) lithium carbonate for two months was monitored for 6 days in the home cage .

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B. Home cage-activity test. Spontaneous locomotor activity of mice chronically fed chow with or without 0.24% (w/w) lithium carbonate for two months was monitored for 6 days in the home cage.


A . Scatter plots showing Venus - fluorescent intensities and the median amplitude of miniature EPSCs ( mEPSC ) in the LA of the AV - KO mice and their littermates ( AV - WT ) . B . Scatter plots showing Venus - fluorescent intensities and the mean frequency of miniature EPSCs in the LA of the AV - KO and their control littermates ( AV - WT ) . Three typical pictures of Venus - positive cells with various fluorescent intensities are shown . Lines in each panel are regression lines showing inverse correlation of the fluorescent intensity with the mEPSC amplitude ( A ) or the mEPSC frequency ( B ) . The black dotted lines are regression lines for AV - WT mice , the red dotted lines are those for AV - KO mice , and black solid lines are those for all cells .

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

A. Scatter plots showing Venus-fluorescent intensities and the median amplitude of miniature EPSCs (mEPSC) in the LA of the AV-KO mice and their littermates (AV-WT). B. Scatter plots showing Venus-fluorescent intensities and the mean frequency of miniature EPSCs in the LA of the AV-KO and their control littermates (AV-WT). Three typical pictures of Venus-positive cells with various fluorescent intensities are shown. Lines in each panel are regression lines showing inverse correlation of the fluorescent intensity with the mEPSC amplitude (A) or the mEPSC frequency (B). The black dotted lines are regression lines for AV-WT mice, the red dotted lines are those for AV-KO mice, and black solid lines are those for all cells.


B . Numbers of Venus - positive neurons in the lateral nucleus ( LA ) and the lateral side of the basal nucleus ( BAl ) of Naive , Unpaired , and Paired groups . Open and filled bars represent AV - WT and AV - KO mice , respectively . Positive neurons of AV - WT was increased only in the Paired group compared to the Naïve and Unpaired groups in the LA ( upper panel ) . AV - KO was increased in the Unpaired as well as the Paired groups compared to the Naïve group Positive neurons both in the Unpaired and Paired AV - KO mice were increased compared to the same groups of the AV - WT mice In the BAl , positive neurons were increased both in the Unpaired and Paired groups compared to the Naïve group in both the AV - WT and AV - KO mice The error bars represent S . E . M . ( n = 5 / group ) .

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

B. Numbers of Venus-positive neurons in the lateral nucleus (LA) and the lateral side of the basal nucleus (BAl) of Naive, Unpaired, and Paired groups. Open and filled bars represent AV-WT and AV-KO mice, respectively. Positive neurons of AV-WT was increased only in the Paired group compared to the Naïve and Unpaired groups in the LA (upper panel). AV-KO was increased in the Unpaired as well as the Paired groups compared to the Naïve group Positive neurons both in the Unpaired and Paired AV-KO mice were increased compared to the same groups of the AV-WT mice In the BAl , positive neurons were increased both in the Unpaired and Paired groups compared to the Naïve group in both the AV-WT and AV-KO mice The error bars represent S.E.M. (n =5/group).


C . Numbers of Venus - positive neurons classified by fluorescence intensities in the LA of the AV - WT and AV - KO mice . Weak positive neurons were increased in the Paired group compared to the Naïve group in AV - WT Both Weak and Middle positive neurons were increased in the Paired group compared to the Naïve group in AV - KO The increment of the Middle Venus - positive neurons was larger in the AV - KO compared to AV -

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C. Numbers of Venus-positive neurons classified by fluorescence intensities in the LA of the AV-WT and AV-KO mice. Weak positive neurons were increased in the Paired group compared to the Naïve group in AV-WT Both Weak and Middle positive neurons were increased in the Paired group compared to the Naïve group in AV-KO The increment of the Middle Venus-positive neurons was larger in the AV-KO compared to AV-WT


Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . LDB2 ( A ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively .

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

Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LDB2 (A) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.


Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . LHX2 ( B ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively .

Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LHX2 (B) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.


Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . LMO2 / 3 / 4 ( C ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively .

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Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LMO2/3/4 (C) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.


Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates , LDB2 vs . SSBP2 / 3 / 4 ( D ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively .

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

Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates , LDB2 vs. SSBP2/3/4 (D) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.


Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . RLIM ( E ) . LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively .

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Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. RLIM (E). LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.


F . Coimmunoprecipitation of endogenous Ssbp3 and Lmo3 with Ldb2 . Immunocomplexes from mouse brain lysate using an antibody against Ldb2 , Lmo3 or Ssbp3 , or a control antibody were analyzed by western blotting using an anti - Ldb2 ( left ) or anti - Ssbp3 ( right ) antibody .

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F. Coimmunoprecipitation of endogenous Ssbp3 and Lmo3 with Ldb2. Immunocomplexes from mouse brain lysate using an antibody against Ldb2, Lmo3 or Ssbp3, or a control antibody were analyzed by western blotting using an anti-Ldb2 (left) or anti-Ssbp3 (right) antibody.


A . Human neurosphere ( bottom ) induced from iPS cells ( top ) from a healthy control subject .

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A. Human neurosphere (bottom) induced from iPS cells (top) from a healthy control subject.


C . Overlap of ChIP - seq peaks of LDB2 in neurosphere cells and ChIP - seq peaks of EGR1 in human cultured cell lines ECC1 and K562 , at

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C. Overlap of ChIP-seq peaks of LDB2 in neurosphere cells and ChIP-seq peaks of EGR1 in human cultured cell lines ECC1 and K562, at ARC


D . Genome - wide pattern of overlap between the ChIP - seq peaks of LDB2 in neurosphere cells and the ChIP - seq peaks of EGR1 in the ECC1 cell line . Fold enrichment ( log2 ) of ChIP - seq reads against the input reads were calculated for each ChIP - seq peak region ( summit ± 1 , 000 bp ) in each category ( neurosphere specific , common , and ECC1 specific ) and displayed in a heatmap by ngs .

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D. Genome-wide pattern of overlap between the ChIP-seq peaks of LDB2 in neurosphere cells and the ChIP-seq peaks of EGR1 in the ECC1 cell line. Fold enrichment (log2) of ChIP-seq reads against the input reads were calculated for each ChIP-seq peak region (summit ± 1,000 bp) in each category (neurosphere specific, common, and ECC1 specific) and displayed in a heatmap by ngs.plot


E . Annotation of ChIP - seq peaks . ChIP - seq peaks of LDB2 in the neurosphere cells ( all , neurosphere specific , and common were analyzed by the PAVIS

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E. Annotation of ChIP-seq peaks. ChIP-seq peaks of LDB2 in the neurosphere cells (all, neurosphere specific, and common were analyzed by the PAVIS program


Representative false colour images of leaf surface temperature captured by a thermal camera from WT , stp1 - 1 , stp4 - 1 , stp1stp4 , stp13 , stp1stp13 and stp4stp13 plants .

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Representative false colour images of leaf surface temperature captured by a thermal camera from WT, stp1-1, stp4-1, stp1stp4, stp13, stp1stp13 and stp4stp13 plants.


Normalized leaf surface temperature over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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Normalized leaf surface temperature over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Whole - plant recordings of changes in stomatal conductance ( gs ) from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . Data shown are means ± SEM ; n ≥ 3 per genotype .

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Whole-plant recordings of changes in stomatal conductance (gs) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.


Whole - plant recordings of changes in stomatal conductance ( gs ) from self - grafted donor lines ( WT / WT , stp1stp4 / stp1stp4 ) and reciprocal grafting of shoot / root ( WT / stp1stp4 , stp1stp4 / WT ) plants . Data shown are means ± SEM ; n = 3 per genotype .

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

Whole-plant recordings of changes in stomatal conductance (gs) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.


Content of soluble sugars in guard cell - enriched epidermal peels of WT and stp1stp4 plants at the end of the night ( EoN ) and after 40 min of illumination with white light at 150 µmol m - 2 s - 1 following the EoN . Data for two independent experiments are shown ; means ± SEM ; n ≥ 7 per genotype and time point .

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Content of soluble sugars in guard cell-enriched epidermal peels of WT and stp1stp4 plants at the end of the night (EoN) and after 40 min of illumination with white light at 150 µmol m-2 s-1 following the EoN. Data for two independent experiments are shown; means ± SEM; n ≥ 7 per genotype and time point.


Representative confocal laser microscopy images of propidium iodide - stained guard cell starch granules of intact leaves of WT , stp1 - 1 , stp4 - 1 , stp1stp4 , suc1 and suc3 plants . Scale bar , 10 µm .

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

Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants. Scale bar, 10 µm.


Starch dynamics in guard cells of intact leaves of WT , stp1 - 1 , stp4 - 1 , stp1stp4 , suc1 and suc3 plants at the end of the night ( EoN ) and after one and three hours of illumination with 150 µmol m - 2 s - 1 of white light . Data for three independent experiments are shown ; means ± SEM ; n = 120 individual guard cells per genotype and time point .

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Starch dynamics in guard cells of intact leaves of WT, stp1-1, stp4-1, stp1stp4, suc1 and suc3 plants at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for three independent experiments are shown; means ± SEM; n = 120 individual guard cells per genotype and time point.


Representative false colour images of photosystem II ( PSII ) operating efficiency ( ΦPSII ) captured by chlorophyll fluorescence imaging from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . ΦPSII was measured at a photosynthetically active radiation ( PAR ) of 440 µmol m - 2 s - 1 .

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

Representative false colour images of photosystem II (PSII) operating efficiency (ΦPSII) captured by chlorophyll fluorescence imaging from WT, stp1-1, stp4-1 and stp1stp4 plants. ΦPSII was measured at a photosynthetically active radiation (PAR) of 440 µmol m-2 s-1.


ΦPSII quantified over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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ΦPSII quantified over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Whole - plant recordings of changes in CO2 assimilation ( A ) from WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants . Data shown are means ± SEM ; n ≥ 3 per genotype .

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

Whole-plant recordings of changes in CO2 assimilation (A) from WT, stp1-1, stp4-1 and stp1stp4 plants. Data shown are means ± SEM; n ≥ 3 per genotype.


Whole - plant recordings of changes in CO2 assimilation ( A ) from self - grafted donor lines ( WT / WT , stp1stp4 / stp1stp4 ) and reciprocal grafting of shoot / root ( WT / stp1stp4 , stp1stp4 / WT ) plants . Data shown are means ± SEM ; n = 3 per genotype .

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

Whole-plant recordings of changes in CO2 assimilation (A) from self-grafted donor lines (WT/WT, stp1stp4/stp1stp4) and reciprocal grafting of shoot/root (WT/stp1stp4, stp1stp4/WT) plants. Data shown are means ± SEM; n = 3 per genotype.


Quantification of leaf glucose ( Glc ) . Data shown are means ± SEM ; n = 8 per genotype and time point . Quantification of leaf fructose ( Fru ) . Data shown are means ± SEM ; n = 8 per genotype and time point . Quantification of leaf sucrose ( Suc ) . Data shown are means ± SEM ; n = 8 per genotype and time point .

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

Quantification of leaf glucose (Glc). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf fructose (Fru). Data shown are means ± SEM; n = 8 per genotype and time point.   Quantification of leaf sucrose (Suc). Data shown are means ± SEM; n = 8 per genotype and time point.


Quantification of leaf starch . Data shown are means ± SEM ; n = 8 per genotype and time point .

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

Quantification of leaf starch. Data shown are means ± SEM; n = 8 per genotype and time point.


Representative Red Green Blue ( RGB ) images of 3 - ( day 0 ) and 4 - week - old ( day 7 ) WT , stp1 - 1 , stp4 - 1 and stp1stp4 plants .

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Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 7) WT, stp1-1, stp4-1 and stp1stp4 plants.


Projected rosette area over the phenotyping period . Data shown are means ± SEM ; n = 10 per genotype and time point .

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Projected rosette area over the phenotyping period. Data shown are means ± SEM; n = 10 per genotype and time point.


Photosynthetic CO2 assimilation ( A ) in dark - adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm . Data shown are means ± SEM ; n = 3 per genotype .

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

Photosynthetic CO2 assimilation (A) in dark-adapted WT and stp1stp4 plants in response to a step increase in CO2 concentrations from 0 to 1000 ppm. Data shown are means ± SEM; n=3 per genotype.


Quantification of leaf glucose ( Glc ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point .

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

Quantification of leaf glucose (Glc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


Quantification of leaf fructose ( Fru ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point . Quantification of leaf sucrose ( Suc ) . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point . Quantification of leaf starch . Data for two independent experiments are shown ; means ± SEM ; n ≥ 17 per genotype and time point .

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

Quantification of leaf fructose (Fru). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf sucrose (Suc). Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.   Quantification of leaf starch. Data for two independent experiments are shown; means ± SEM; n ≥ 17 per genotype and time point.


Representative Red Green Blue ( RGB ) images of 3 - ( day 0 ) and 4 - week - old ( day 6 ) WT and stp1stp4 plants grown under 600 ppm CO2 .

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

Representative Red Green Blue (RGB) images of 3-(day 0) and 4-week-old (day 6) WT and stp1stp4 plants grown under 600 ppm CO2.


Projected rosette area over the phenotyping period . Data shown are from two independent experiments ; means ± SEM ; n ≥ 24 per genotype and time point .

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Projected rosette area over the phenotyping period. Data shown are from two independent experiments; means ± SEM; n ≥ 24 per genotype and time point.


Representative confocal laser microscopy images of propidium iodide - stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 . Scale bar , 10 µm .

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

Representative confocal laser microscopy images of propidium iodide-stained guard cell starch granules of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2. Scale bar, 10 µm.


Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night ( EoN ) and after one and three hours of illumination with 150 µmol m - 2 s - 1 of white light . Data for two independent experiments are shown ; means ± SEM ; n = 80 individual guard cells per genotype and time point .

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

Starch dynamics in guard cells of intact leaves of WT and stp1stp4 plants grown under 600 ppm CO2 at the end of the night (EoN) and after one and three hours of illumination with 150 µmol m-2 s-1 of white light. Data for two independent experiments are shown; means ± SEM; n = 80 individual guard cells per genotype and time point.