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 .

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 .

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 .

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 .

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).


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 .

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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 ) .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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 .

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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 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 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 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

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 O O O O O O O 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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


A , Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA , or 5 nM Actinomycin D ( ActD ) . GAPDH served as a loading control .

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

A, Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA, or 5 nM Actinomycin D (ActD). GAPDH served as a loading control.


B , HCT116 cells were treated for 24 hrs with vehicle alone ( - ) , 1 µM MPA , 10 µM MPA , or the combination of 10 µM MPA with 400 µM guanosine . Guanylate nucleotides levels were measured by LC - MS , and normalized to protein content . Mean ± SEM is representative of 3 independent experiments carried out in triplicate . Data information data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

O O 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HCT116 cells were treated for 24 hrs with vehicle alone (-), 1 µM MPA, 10 µM MPA, or the combination of 10 µM MPA with 400 µM guanosine. Guanylate nucleotides levels were measured by LC-MS, and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate. Data information data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


Parental HCT116 cells expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) and either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in the absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . mRNAs of IMPDH1 and IMPDH2 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate and normalized to β - actin mRNA ( C ) . Data information : data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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

Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


Parental HCT116 cells expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) and either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in the absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . Whole cell extracts were analyzed on Western blots with the indicated antibodies . GAPDH was used as a loading control . Quantification of band intensity of p53 and p21 is shown . Mean ± SD is representative of four independent experiments . ( right panel ) ( D ) . Data information : data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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

Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). Whole cell extracts were analyzed on Western blots with the indicated antibodies. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 is shown. Mean ± SD is representative of four independent experiments. (right panel) (D). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


E , Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA , in presence of dimethyl sulfoxide ( DMSO ) or 400 µM guanosine . GAPDH was used as a loading control .

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

E, Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA, in presence of dimethyl sulfoxide (DMSO) or 400 µM guanosine. GAPDH was used as a loading control.


A , HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone ( - ) or the indicated concentration of MPA for 24 hrs . The levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 and p21 of four independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone (-) or the indicated concentration of MPA for 24 hrs. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 of four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


B , HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 of at least four independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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

B, HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


C , HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence ( - ) or the presence ( + ) of the combination of 10 µM of the ATR inhibitor VE - 821 ( ATRi ) and 10 µM the ATM inhibitor KU - 55933 ( ATMi ) , followed by addition of the vehicle alone ( - ) or MPA 10 µM for additional 24 hrs . The levels of p53 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 of at least two independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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

C, HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence (-) or the presence (+) of the combination of 10 µM of the ATR inhibitor VE-821 (ATRi) and 10 µM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone (-) or MPA 10 µM for additional 24 hrs. The levels of p53 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least two independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


A , HCT116 cells were treated with either vehicle alone ( - ) , 1 µM MPA , 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA . Data information : , data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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

A, HCT116 cells were treated with either vehicle alone (-), 1 µM MPA, 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA. Data information: , data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


B , Autoradiogram ( upper panel ) and Ethidium Bromide ( EtBr ) - stained agarose gel ( lower panel ) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section .

O O O O O O O O O O O O O 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, Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section.


C , Autoradiogram ( upper panel ) and EtBr - stained TBE - urea polyacrylamide gel ( lower panel ) of 5S , 5 . 8S rRNA and tRNAs in

O O O O 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

C, Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysates


HCT116 cells were treated for 24 hrs . Cell lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA and immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or the IgG control ( IgG ) . D , Levels of HDM2 , RPL5 , RPL11 , and the IP negative control mTOR in the whole cell extract ( WCE ) , the post - ribosomal lysates ( INPUT , left panel ) and in the HDM2 immunoprecipitated fraction ( right panel ) were analyzed on Western blots . The results are representative of two independent experiments . Quantification of band intensity of HDM2 , RPL5 and RPL11 of at least two independent experiments is shown ( lower right panel ) .

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

HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). D, Levels of HDM2, RPL5, RPL11, and the IP negative control mTOR in the whole cell extract (WCE), the post-ribosomal lysates (INPUT, left panel) and in the HDM2 immunoprecipitated fraction (right panel) were analyzed on Western blots. The results are representative of two independent experiments. Quantification of band intensity of HDM2, RPL5 and RPL11 of at least two independent experiments is shown (lower right panel).


HCT116 cells were treated for 24 hrs . Cell lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA and immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or the IgG control ( IgG ) . E , the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA . Data are normalized and presented as relative to untreated cells immunoprecipitated with anti - HDM2 . The results are representative of three independent experiments carried out in triplicates . Data information : data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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

HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). E, the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA. Data are normalized and presented as relative to untreated cells immunoprecipitated with anti-HDM2. The results are representative of three independent experiments carried out in triplicates. Data information: data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


F , HCT116 cells were pretreated with vehicle alone ( - ) , 1 µM , or 10 µM MPA for 24 hrs , and the levels of p53 and p21 were followed on Western blots in presence of 100 µg / mL cycloheximide ( CHX ) for the indicated time , in the continued presence of the treatment . GAPDH was used as a loading control . Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment .

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

F, HCT116 cells were pretreated with vehicle alone (-), 1 µM, or 10 µM MPA for 24 hrs, and the levels of p53 and p21 were followed on Western blots in presence of 100 µg/mL cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment.


G , HCT116 cells were pretreated as in ( F ) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated . The levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel . Data information : In panels data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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

G, HCT116 cells were pretreated as in (F) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel. Data information: In panels data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


A , HCT116 were treated as indicated for 24 hrs , subjected to propidium iodide staining and analyzed by fluorescence - activated cell sorting ( FACS ) . Data are representative of at least three independent experiments .

O O O O O O O O O O O O O O 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, HCT116 were treated as indicated for 24 hrs, subjected to propidium iodide staining and analyzed by fluorescence-activated cell sorting (FACS). Data are representative of at least three independent experiments.


B , Colon adenocarcinoma cell lines HCT116 ( circles ) , LoVo ( squares ) , LS174 ( triangles ) , or RKO ( inversed triangles ) were treated with the vehicle alone ( - ) , 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis . Data are shown as the fold induction of the cell number in G0 / G1 phases ( blue ) , S phase ( red ) , and G2 / M phases upon treatment with 1 µM MPA ( dark ) or 10 µM MPA ( clear ) over vehicle treated cells .

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

B, Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 µM MPA (dark) or 10 µM MPA (clear) over vehicle treated cells.


C , IdU track length distribution , and distribution of long fork / short fork IdU track length ratios ( insert ) . IdU track length of at least 300 fibers was measured for each condition . For fork symmetry analyses , IdU track length was measure in at least 38 bidirectional forks ( see methods ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis test of the means . D , Representative images from ( C ) .

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

C, IdU track length distribution, and distribution of long fork / short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see methods). ***p<0.001, ****p<0.0001 by Kruskal-Wallis test of the means. D, Representative images from (C).


E , HCT116 cells , pre - incubated with 10 µM BrdU for 48 hrs were treated with Vehicle ( - ) , 10µM MPA for 6 or 24 hrs , or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation ( see Method ) . Cells were immunostained for native BrDU ( green ) , or RPA ( red ) . DNA was counterstained with DAPI ( blue ) . One representative z - confocal stack is shown per condition . Scale bars correspond to 5 μm .

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

E, HCT116 cells, pre-incubated with 10 µM BrdU for 48 hrs were treated with Vehicle (-), 10µM MPA for 6 or 24 hrs, or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation (see Method). Cells were immunostained for native BrDU (green), or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 μm.


F , HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins . α - tubulin was used as a loading control .

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

F, HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins. α-tubulin was used as a loading control.


A , HCT116 cells were treated with either the vehicle alone ( - ) , 1 µM MPA or 10 µM MPA for 24 hrs , before p21 immuno - labeling , propidium iodide staining and FACS analysis . Cell cycle profiles of the total cell population ( upper panel , left ) , the total intensity of p21 and propidium iodide were determined in 20 , 000 cells and plotted in a scatter diagram ( lower panel , left ) . The specific cell cycle profiles are shown of p21high ( upper panel , right ) and p21low ( lower panel , right ) cell populations . Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels .

O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HCT116 cells were treated with either the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs, before p21 immuno-labeling, propidium iodide staining and FACS analysis. Cell cycle profiles of the total cell population (upper panel, left), the total intensity of p21 and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels.


B , HCT116 p21 - / - isogenic cell lines were transfected for 24 hrs with an empty plasmid ( EV ) or a plasmid encoding a p21wt cDNA ( p21 - OE ) , then cells were serum - deprived for 16 hrs before the re - addition of serum for additional 24 hrs in absence or presence of 10 µM MPA . Cells were subjected to p21 immunolabeling , propidium iodide staining and FACS analysis . The results are representative of two independent experiments .

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

B, HCT116 p21-/- isogenic cell lines were transfected for 24 hrs with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), then cells were serum-deprived for 16 hrs before the re-addition of serum for additional 24 hrs in absence or presence of 10 µM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining and FACS analysis. The results are representative of two independent experiments.


A , HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in ( Fig 5B ) . Here are shown the cell cycle profiles after 16 hrs of serum deprivation ( top panels ) , or after an additional 24 hrs of serum re - addition in absence ( middle panels ) , or presence of 10 µM MPA ( bottom panels ) . The results are representative of at least three independent experiments .

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

A, HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in (Fig 5B). Here are shown the cell cycle profiles after 16 hrs of serum deprivation (top panels), or after an additional 24 hrs of serum re-addition in absence (middle panels), or presence of 10 µM MPA (bottom panels). The results are representative of at least three independent experiments.


B , HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs . The levels of γ - H2AX were analyzed on Western blots . GAPDH was used as a loading control .

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

B, HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs. The levels of γ-H2AX were analyzed on Western blots. GAPDH was used as a loading control.


Statistical analysis of the immunostaining of γ - H2AX ( C ) in HCT116 cells treated Total nuclear intensities were obtained on > 6000 ( γ - H2AX ) individual cells for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis test . A . U . Arbitrary Units .

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

Statistical analysis of the immunostaining of γ-H2AX (C) in HCT116 cells treated Total nuclear intensities were obtained on >6000 (γ-H2AX) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.


Statistical analysis of the immunostaining of 53BP1 ( D ) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 ( 53BP1 ) individual cells for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis test . A . U . Arbitrary Units .

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

Statistical analysis of the immunostaining of 53BP1 (D) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 (53BP1) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.


E , Representative images cells labeled with γ - H2AX ( green ) , 53BP1 ( red ) and DAPI ( blue ) . Scale bars correspond to 10

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E, Representative images cells labeled with γ-H2AX (green), 53BP1 (red) and DAPI (blue). Scale bars correspond to 10 μm


F , HCT116 cells treated were analyzed by the comet assay . The percentage of tail DNA / total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates . * * * * p < 0 . 0001 by Kruskal - Wallis

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

F, HCT116 cells treated were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****p<0.0001 by Kruskal-Wallis test


( A - C ) Western blot analysis of THP - 1 cells infected with ( A ) HSV - 1 at MOI 5 for 3 h , 6 h , 12 h , 18 h , and 24 h ( B ) CHIKV at MOI 5 for 3 h , 6 h , 12 h , 18 h , and 24 h ( C ) JEV at MOI 5 for 3 h , 6 h , 12 h , and 24 h .

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

(A-C) Western blot analysis of THP-1 cells infected with (A) HSV-1 at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (B) CHIKV at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (C) JEV at MOI 5 for 3 h, 6 h, 12 h, and 24 h.


( I - L ) Control and IRGM knockdown THP - 1 IFN reporter cells were treated with ( I ) Poly I : C ( 1 μg / ml ) for 8 h , 12 h and 24 h or ( J ) Poly dA : dT ( 1 μg / ml ) for 12 h and 24 h or ( K ) IFN - β ( 500 ng / ml ) for 8 h and 12 h or ( L ) cGAMP ( 1 μg / ml ) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc

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

(I-L) Control and IRGM knockdown THP-1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN-β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent


( M - P ) Control and IRGM knockdown THP - 1 cells were untreated and treated with Poly I : C ( 1 μg / ml ) for 8 h and the total RNA was subjected to qRT PCR with primers of ( M ) MX2 ( N ) ISG15 ( O ) OAS1 and ( P ) IFN - β . ( Q - T ) Control and IRGM knockdown THP - 1 cells were untreated and treated with Poly dA : dT ( 1 μg / ml ) for 8 h and the total RNA was subjected to qRT PCR with primers of ( Q ) MX2 ( R ) ISG15 ( S ) OAS1 and ( T ) IFN - β .

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

(M-P) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN-β. (Q-T) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN-β.


( D ) RNA was isolated from irgm1 + / + and irgm1 - / - BMDMs and subjected to qRT - PCR with indicated viral restriction factor

O O O O 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) RNA was isolated from irgm1+/+ and irgm1-/- BMDMs and subjected to qRT-PCR with indicated viral restriction factor genes


( H - J ) RNA isolated from control and IRGM knockdown HT - 29 cells and subjected to qRT - PCR with indicated genes of ( H ) Immunoproteasome complex ( I ) TAP complex ( J ) human leukocyte antigen ( HLA ) system .

O O O O O O O O O O O O O O O O O O O O O O O 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-J) RNA isolated from control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system.


( L , M ) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP - 1 cells treated with DQ - OVA ( green ) ( 10 µg / ml , 30 min ) . Scale Bar , 10 µm . ( M ) Graph depicts percentage of control and IRGM knockdown THP - 1 cells with DQ - OVA puncta '

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, M) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP-1 cells treated with DQ-OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP-1 cells with DQ-OVA puncta's


( P ) Representative confocal images of H - 2Kb - SIINFEKL ( red ) on the surface of Irgm1 + / + and irgm1 - / - mouse BMDMs treated with OVA ( 2 mg / ml , 3 h ) , Scale bar 5 µm or 8 µm ( as indicated ) .

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

(P) Representative confocal images of H-2Kb-SIINFEKL (red) on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h), Scale bar 5 µm or 8 µm (as indicated).


( C ) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(C) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( F ) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV - 1 ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(F) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV-1 (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( I ) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV ( MOI 1 , 24 h ) infected HT - 29 control and IRGM knockdown cells .

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(I) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( L ) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV ( MOI 0 . 3 , 16 h ) infected HuH7 control and IRGM knockdown cells .

(L) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV (MOI 0.3, 16 h) infected HuH7 control and IRGM knockdown cells.


( O ) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV ( MOI 5 , 48 h ) infected Huh7 control and IRGM knockdown cells .

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

(O) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells.


( S ) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS - CoV2 ( MOI 1 , 24 h ) infected THP - 1 control and IRGM knockdown cells .

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

(S) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS-CoV2 (MOI 1, 24 h) infected THP-1 control and IRGM knockdown cells.


( D ) Kaplan - Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1 + / + and irgm1 - / - neonates during the course of

O O O O O O 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) Kaplan-Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1+/+ and irgm1-/- neonates during the course of infection


( H , I ) Total RNA isolated from muscles and brains of mock and CHIKV infected ( MOI 1x107 PFU / mouse ) ( 6 dpi ) irgm1 + / + and irgm1 - / - mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles .

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

(H, I) Total RNA isolated from muscles and brains of mock and CHIKV infected (MOI 1x107 PFU/mouse) (6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles.


( A - D ) Control and si - IRGM transfected THP - 1 IFN reporter cells were kept uninfected ( Mock ) or infected with ( A ) JEV ( MOI 5 ) or ( B ) CHIKV ( MOI 5 ) or ( C ) HSV - 1 ( MOI 2 . 5 ) or ( D ) VSV - eGFP ( MOI 1 ) and the supernatant collected 8 hpi were subjected to luciferase assay . The graphs depict fold change in interferon response .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Control and si-IRGM transfected THP-1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV-1 (MOI 2.5) or (D) VSV-eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response.


( L ) Western blot analysis with cell lysates of mock and CHIKV ( MOI 5 , 24 h ) infected control and IRGM knockdown HT - 29 cells and probed with the indicated antibodies . ( M ) Western blot analysis with cell lysates of mock and JEV ( MOI 5 , 24 h ) infected control and si - IRGM transfected HT - 29 cells and probed with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT-29 cells and probed with the indicated antibodies. (M) Western blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si-IRGM transfected HT-29 cells and probed with the indicated antibodies.


( Q ) Western blot analysis with cell lysates of mock and CHIKV ( MOI 1 , 24 h ) infected control and IRGM knockdown HT - 29 cells transfected with indicated siRNA and probed with the indicated antibodies .

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

(Q) Western blot analysis with cell lysates of mock and CHIKV (MOI 1, 24 h) infected control and IRGM knockdown HT-29 cells transfected with indicated siRNA and probed with the indicated antibodies.


A Representative immunofluorescence for PH3 ( green ) , combined with DAPI staining ( white ) , of E14 . 5 wildtype ( WT ) and 11B mouse dorsolateral neocortex rostrally . B Representative immunofluorescence for pVim ( yellow ) , combined with DAPI staining ( white ) , of E14 . 5 WT and 11B mouse dorsolateral neocortex rostrally . Boxes ( 50 µm x 65 µm ) in ( B ) indicate areas that are shown at higher magnification in ( B ' ) . Arrow , pVim + BP with basal process ( mitotic bRG ) ; notched arrowheads , basal process ; arrowhead , pVim + BP without basal process ( mitotic bIP ) . C - E Quantification of PH3 + APs ( C ) , PH3 + BPs ( D ) and pVim + bRG ( E left ) and bIPs ( E right ) in a 200 µm - wide field of E14 . 5 WT ( white ) and 11B ( black ) mouse dorsolateral neocortex rostrally . Note the difference in ordinate scales in ( E ) . F Data information : Images are single optical sections Scale bars , 50 µm 10 µm Data are the mean of 6 - 19 ( WT ) and 6 - 26 ( 11B ) littermate embryos , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0269 ( D ) ; P = 0 . 0302 ( E , left ) ; P = 0 . 0139 ( E , right )

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A Representative immunofluorescence for PH3 (green), combined with DAPI staining (white), of E14.5 wildtype (WT) and 11B mouse dorsolateral neocortex rostrally. B Representative immunofluorescence for pVim (yellow), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (50 µm x 65 µm) in (B) indicate areas that are shown at higher magnification in (B'). Arrow, pVim+ BP with basal process (mitotic bRG); notched arrowheads, basal process; arrowhead, pVim+ BP without basal process (mitotic bIP). C-E Quantification of PH3+ APs (C), PH3+ BPs (D) and pVim+ bRG (E left) and bIPs (E right) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. Note the difference in ordinate scales in (E). F Data information: Images are single optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0269 (D); P = 0.0302 (E, left); P = 0.0139 (E, right)


F Representative immunofluorescence for Pax6 ( cyan ) and Tbr2 ( magenta ) , combined with DAPI staining ( white ) , of E14 . 5 WT and 11B mouse dorsolateral neocortex rostrally . Boxes ( 25 µm x 25 µm ) in ( F ) indicate areas that are shown at higher magnification in ( F ' ) . Outlined by dashed white lines , Pax6 + & Tbr2 + BP ; outlined by solid white lines , Pax6 + & Tbr2 - BP . G - K Quantification of Pax6 + & Tbr2 - cells in VZ ( G ) , Pax6 + & Tbr2 + cells in VZ ( H ) , Pax6 + & Tbr2 - cells in SVZ and IZ ( I ) , Pax6 + & Tbr2 + cells in SVZ and IZ ( J ) and Pax6 - & Tbr2 + cells in SVZ and IZ ( K ) in a 200 µm - wide field of E14 . 5 WT ( white ) and 11B ( black ) mouse dorsolateral neocortex rostrally . L Data information : Images are Z projections of 4 optical sections Scale bars , 50 µm 10 µm Data are the mean of 6 - 19 ( WT ) and 6 - 26 ( 11B ) littermate embryos , which were derived from 3 - 9 separate litters . Error bars indicate SD , two - tailed unpaired Student ' s t - test , * P < 0 . 05 ; * * P < 0 . 01 P = 0 . 0281 ( I ) ; P = 0 . 0088 ( J ) ; P = 0 . 0014 ( K )

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F Representative immunofluorescence for Pax6 (cyan) and Tbr2 (magenta), combined with DAPI staining (white), of E14.5 WT and 11B mouse dorsolateral neocortex rostrally. Boxes (25 µm x 25 µm) in (F) indicate areas that are shown at higher magnification in (F'). Outlined by dashed white lines, Pax6+ & Tbr2+ BP; outlined by solid white lines, Pax6+ & Tbr2- BP. G-K Quantification of Pax6+ & Tbr2- cells in VZ (G), Pax6+ & Tbr2+ cells in VZ (H), Pax6+ & Tbr2- cells in SVZ and IZ (I), Pax6+ & Tbr2+ cells in SVZ and IZ (J) and Pax6- & Tbr2+ cells in SVZ and IZ (K) in a 200 µm-wide field of E14.5 WT (white) and 11B (black) mouse dorsolateral neocortex rostrally. L Data information: Images are Z projections of 4 optical sections Scale bars, 50 µm 10 µm Data are the mean of 6-19 (WT) and 6-26 (11B) littermate embryos, which were derived from 3-9 separate litters. Error bars indicate SD, two-tailed unpaired Student's t-test, * P < 0.05; ** P < 0.01 P = 0.0281 (I); P = 0.0088 (J); P = 0.0014 (K)