( C , D ) Confocal z - projections of adult DIP - δT2A - Gal4 / 1 - 119 trans - heterozygous mutant ( D ) or DIP - δ1 - 119 / + heterozygous ( C ) brains , which express MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ; green ) , in which DIP - δ - Gal4 either drives expression or UAS - DIP - α ( D ) or not ( C ) . Grayscale panels represent single channels , as indicated . γ - lobe is outlined 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Confocal z-projections of adult DIP-δT2A-Gal4/1-119 trans-heterozygous mutant (D) or DIP-δ1-119/+ heterozygous (C) brains, which express MiMIC mediated Dpr12GFSTF (Dpr12-GFP; green), in which DIP-δ-Gal4 either drives expression or UAS-DIP-α (D) or not (C). Grayscale panels represent single channels, as indicated. The γ-lobe is outlined in white.


( E , F ) Confocal z - projections of adult dpr12Δ50 - 81 homozygous mutant brains , in which DIP - δ - Gal4 drives expression of either UAS - mtdT ( E ) or UAS - DIP - α - T2A - tdT ( F ; expected to induce expression of DIP - α as well as tdT encoded by a polycistronic message ) . In orange are longitudinal sections across γ - lobe at indicated location . γ - lobe is outlined in , as determined by FasII ( grey in E ' - F ' ) . Cyan is tdT within DIP - δ + . Grey boxes below describe relevant components within PAM - DANs γ - KCs .

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(E, F) Confocal z-projections of adult dpr12Δ50-81 homozygous mutant brains, in which DIP-δ-Gal4 drives expression of either UAS-mtdT (E) or UAS-DIP-α-T2A-tdT (F; expected to induce expression of DIP-α as well as tdT encoded by a polycistronic message). In orange are longitudinal sections across the γ-lobe at the indicated location. The γ-lobe is outlined in white, as determined by FasII staining (grey in E'-F'). Cyan is tdT within DIP-δ+ cells. Grey boxes below the images describe the relevant components within PAM-DANs and γ-KCs.


A . Stills depicting actin flow in extracellular RH , myoA KO adf KD expressing Cb EmeraldFP . Continuous F - actin flow can be seen from apical tip , to Golgi region towards posterior pole of . No difference can be observed between RH myoA KO . For adf KD , flow is completely abrogated . Red arrowhead marks Golgi region , while yellow arrowhead marks apical nucleation centre . B . Skeletonisation processing of ( A ) depicting areas where F - actin dynamics / flow is prevalent . signals for F - actin form a continuous , dynamic network that connects apical posterior pole of . Red arrowhead marks Golgi region nucleation centre , while yellow arrowhead marks apical nucleation centre . represent a projection of frames for each timepoint to provide a better overview of dynamics of F - actin . C . Kymograph of ( A ) ( B ) . kymograph was generated by tracing a in middle of ' s ( red ) . colour - coded kymograph represents forward movement ( red ) , backwards movement ( green ) static F - actin ( blue ) . results demonstrate continuous exchange of F - actin from apical to posterior pole of within cytosol of . This exchange is similar in RH myoA KO , while completely abolished in case of adf KD . Data information : Numbers indicate minutes : seconds ; Scale bars represent 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Stills depicting actin flow in extracellular RH, myoA KO and adf KD parasites expressing Cb EmeraldFP. Continuous F-actin flow can be seen from the apical tip, to the Golgi region towards the posterior pole of the parasite. No difference can be observed between RH and myoA KO. For adf KD, the flow is completely abrogated. Red arrowhead marks the Golgi region, while the yellow arrowhead marks the apical nucleation centre. B. Skeletonisation processing of (A) depicting areas where F-actin dynamics/flow is prevalent. Individual signals for F-actin form a continuous, dynamic network that connects apical and posterior pole of the parasite. Red arrowhead marks the Golgi region nucleation centre, while the yellow arrowhead marks the apical nucleation centre. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin. C. Kymograph analysis of (A) and (B). The kymograph was generated by tracing a line in the middle of the parasite's body (red line). The colour-coded kymograph represents forward movement (red), backwards movement (green) and static F-actin (blue). The results demonstrate continuous exchange of F-actin from the apical to the posterior pole of the parasite within the cytosol of the parasite. This cytosolic exchange is similar in RH and myoA KO parasites, while completely abolished in the case of adf KD. Data information: Numbers indicate minutes:seconds; and Scale bars represent 5 µm.


D . Top three rows : expressing Cb EmeraldFP along with myoA - SNAP before after Ca2 + - Ionophore ( A23187 ) or BIPPO treatment . were also treated with 2 µM of Cytochalasin D or DMSO ( as control ) for 30 min . After addition of A23187 , preferential relocalisation of actin can be observed at apical tip , while addition of BIPPO caused F - actin accumulation at basal end . 4th row : myoAKO expressing Cb - EmeraldFP before after treatment with A23187 BIPPO . While no apical or basal accumulation of F - actin is observed , some peripheral location of F - actin occurs in presence of A23187 or BIPPO , preferentially in apical half of . Bottom row : adf cKD expressing Cb - EmeraldFP before after treatment with Ca2 + Ionophore or BIPPO . No apparent change in F - actin localisation can be seen upon treatment . E . Skeletonization of movies shown in D . Before addition of A23187 or BIPPO , RH myoA KO behave similar with no accumulation of F - actin at apical tip . After treatment with calcium ionophore A23187 , preferential accumulation of F - actin at apical tip can be observed for RH ( red arrowhead ) , while BIPPO presented preferential accumulation in basal end ( red arrowhead ) . In myoA KO , some relocalisation of actin is observed at periphery of ( yellow arrowhead ) . In case of adf cKD , no relocalisation occurs . represent a projection of frames for each timepoint to provide a better overview of dynamics of F - actin .

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

D. Top three rows: Parasites expressing Cb EmeraldFP along with myoA-SNAP before and after Ca2+-Ionophore (A23187) or BIPPO treatment. The parasites were also treated with 2 µM of Cytochalasin D or DMSO (as control) for 30 min. After addition of A23187, preferential relocalisation of actin can be observed at the apical tip, while addition of BIPPO caused F-actin accumulation at the basal end. 4th row: myoAKO parasites expressing Cb-EmeraldFP before and after treatment with A23187 and BIPPO. While no apical or basal accumulation of F-actin is observed, some peripheral location of F-actin occurs in presence of A23187 or BIPPO, preferentially in the apical half of the parasite. Bottom row: adf cKD parasites expressing Cb-EmeraldFP before and after treatment with Ca2+ Ionophore or BIPPO. No apparent change in F-actin localisation can be seen upon treatment. E. Skeletonization of movies shown in D. Before addition of A23187 or BIPPO, RH and myoA KO parasites behave similar with no accumulation of F-actin at the apical tip. After treatment with calcium ionophore A23187, preferential accumulation of F-actin at the apical tip can be observed for RH (red arrowhead), while BIPPO presented preferential accumulation in the basal end (red arrowhead). In myoA KO, some relocalisation of actin is observed at the periphery of the parasite (yellow arrowhead). In the case of adf cKD, no relocalisation occurs. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin.


F . of actin accumulation before after treatment with Ca2 + Ionophore or BIPPO . were counted for F - actin accumulation after adding Ca2 + Ionophore or BIPPO . Numbers were generated by counting total number of then number of with actin accumulation on apical or basal tip . A minimum of 200 were counted upon 3 biological replicates . Two - way ANOVA was used for statistical Tukey ' s multiple comparisons . * * * * p value < 0 . 0001 . Error bars represent standard deviation .

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 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

F. Quantification of actin accumulation before and after treatment with Ca2+ Ionophore or BIPPO. The parasites were counted for F-actin accumulation after adding Ca2+ Ionophore or BIPPO. Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip. A minimum of 200 parasites were counted upon 3 biological replicates. Two-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. Error bars represent standard deviation.


A . Wild Type RH mid - invasion . Tight junction was done by allowing freshly egressed to invade for 5 minutes , fixed with 4 % PFA before labelling TJ with anti - Ron2 ( yellow ) DAPI ( magenta ) for . arrow points at invasion direction .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Wild Type RH parasites mid-invasion. Tight junction assay was done by allowing freshly egressed parasite to invade for 5 minutes, fixed with 4% PFA before labelling the TJ with anti-Ron2 (yellow) and DAPI (magenta) for nuclear staining. White arrow points at invasion direction.


B . Graph showing deformation of whole its nucleus between invading non - invading . 100 found mid - invasion were counted in triplicate compared to extracellular , freshly egressed . One - way ANOVA was used for statistical . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . Scale bars represent 5 µm .

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B. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. 100 parasites found mid-invasion were counted in triplicate and compared to extracellular, freshly egressed parasites. One-way ANOVA was used for statistical analysis. **** p value <0.0001. Data information: Error bars represent standard deviation. and Scale bars represent 5 µm.


C . Graph showing deformation of whole its nucleus between invading non - invading . Indicated were analysed . For adf cKD induced non - induced conditions were compared , as indicated . 30 were counted for each condition in triplicate . One - way ANOVA was used for statistical Tukey ' s multiple comparisons . * * * * p value < 0 . 0001 . * * * p value 0 . 0009 . Data information : Error bars represent standard deviation . ; Scale bars represent 5 µm .

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C. Graph showing deformation of the whole parasite and its nucleus between invading and non-invading parasites. Indicated parasites were analysed. For adf cKD induced and non-induced conditions were compared, as indicated. 30 parasites were counted for each condition in triplicate. One-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. *** p value 0.0009. Data information: Error bars represent standard deviation. ; and Scale bars represent 5 µm.


D . Time - lapse microscopy of indicated invading HFFs . Time was determined from onset of invasion . arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows apical pole of . Numbers indicate minutes :

O O O O O O O O O O O O O O O O O O O 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. Time-lapse microscopy analysis of indicated parasites invading HFFs cells. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds


E . Speed profiles of invading relative displacement of apical tip over time allowed speed calculations . stages were named in regard to invasion step ( Penetration initiation , Speed decrease finalisation ) . Invasion was analysed with Icy Image Processing Software ( Pasteur Institut ) using Manual Tracking plugin by analysing each movie 10 times averaging

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Speed profiles of invading parasites The relative displacement of the apical tip over time allowed speed calculations. Individual stages were named in regard to the invasion step (Penetration initiation, Speed decrease and finalisation). Invasion was analysed with Icy Image Processing Software (Pasteur Institut) using the Manual Tracking plugin by analysing each movie 10 times and averaging the measurements


F . Time - lapse demonstrating abortive invasion of indicated . Time was determined from onset of invasion . arrow points at invasion direction ; yellow arrow points at tight junction ; red dot follows apical pole of . Numbers indicate minutes :

F. Time-lapse analysis demonstrating abortive invasion of indicated parasites. Time was determined from the onset of invasion. White arrow points at invasion direction; yellow arrow points at tight junction; red dot follows the apical pole of the parasite. Numbers indicate minutes:seconds


A . Time - lapse depicting invading RH adfcKD expressing Cb - EmeraldFP . A F - actin ring can be observed at TJ in majority of cases ( red arrowheads , first panel ) . Invasion can also occur without formation of an F - actin ring ( second panel ) . During capped invasion , an F - actin ring is formed ( third panel ) . Invasion of adf cKD occurs without formation of an F - actin ring ( fourth panel ) . F - actin accumulation is observed at posterior pole of in cases ( orange arrows ) . Green segmented lines depict that was measured for generation of plot profiles shown in ( B ) . Yellow purple dotted lines indicate extra - intracellular part of respectively . B . plot profiles of movies shown in ( A ) . Depicted plots correlate to half - invaded . In case of F - actin ring formation , two distinct peaks are detected , correlating to F - actin ring at TJ ( red arrowhead ) posterior pole of ( orange arrow ) . In case of no F - actin ring , no accumulation at TJ can be observed ; only peak correlating to posterior F - actin can be detected . For adf cKD , only one peak can be seen corresponding to posterior pole of . C . of F - actin accumulation at TJ for RH Cb - EmeraldFP as detected in time lapse . values are expressed as . 28 total invasion events were captured across three different biological replicates . Two - unpaired t - was performed . * * * * p value < 0 . 0001 . Data information : Error bars represent standard deviation . arrows point to direction of invasion . Scale bars represent 5 µm . * * * * p value < 0 . 0001 .

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

A. Time-lapse analysis depicting invading RH adfcKD parasites expressing Cb-EmeraldFP. A F-actin ring can be observed at the TJ in the majority of cases (red arrowheads, first panel). Invasion can also occur without formation of an F-actin ring (second panel). During capped invasion, an F-actin ring is formed (third panel). Invasion of adf cKD parasites occurs without formation of an F-actin ring (fourth panel). F-actin accumulation is observed at the posterior pole of the parasite in all cases (orange arrows). Green segmented lines depict the area that was measured for generation of intensity plot profiles shown in (B). Yellow and purple dotted lines indicate the extra- and intracellular part of the parasite respectively. B. Intensity plot profiles of the movies shown in (A). Depicted plots correlate to half-invaded parasites. In the case of F-actin ring formation, two distinct peaks are detected, correlating to the F-actin ring at the TJ (red arrowhead) and the posterior pole of the parasite (orange arrow). In the case of no F-actin ring, no accumulation at the TJ can be observed; only the peak correlating to posterior F-actin can be detected. For adf cKD, only one peak can be seen corresponding to the posterior pole of the parasite. C. Quantification of F-actin accumulation at the TJ for RH Cb-EmeraldFP parasites as detected in time lapse analysis. The values are expressed as percentage. 28 total parasite invasion events were captured across three different biological replicates. Two-tailed unpaired t-test analysis was performed. **** p value <0.0001. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm. ****p value <0.0001.


D . Representative immunofluorescence assays on indicated strains during host penetration . were labelled with indicated antibodies . SAG1 was performed prior to permeabilisation to stain only extracellular portion of . Antibodies against Ron2 were used to stain TJ . Red arrowhead : position of TJ . Orange arrow : posterior pole of . Data information : Error bars represent standard deviation . arrows point to direction of invasion . Scale bars represent 5 µm .

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D. Representative immunofluorescence assays on indicated parasite strains during host cell penetration. Parasites were labelled with indicated antibodies. SAG1 staining was performed prior to permeabilisation to stain only the extracellular portion of the parasite. Antibodies against Ron2 were used to stain the TJ. Red arrowhead: position of the TJ. Orange arrow: posterior pole of the parasite. Data information: Error bars represent standard deviation. White arrows point to direction of invasion. Scale bars represent 5 µm.


E . Tight Junction on fixed samples depicting invasion rates of indicated . While in majority of control F - actin can be detected at junction ( ~ 80 % ) , no junctional ring can be detected in ~ 20 % of invasion events . In contrast , adf cKD , act1 cKO or myoA KO only show an overall invasion rate of ~ 20 % compared to controls . In these cases junctional F - actin accumulation could never be detected . Note that number of total invasion events for adf cKD , act1cKO myoAKO correlates to number of invasion events , seen for RH without F - actin ring formation . Data information : Error bars represent standard deviation . One - way ANOVA was performed for each graph * * * * p value < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Tight Junction assay on fixed samples depicting invasion rates of indicated parasites. While in the majority of control parasites F-actin can be detected at the junction (~80%), no junctional ring can be detected in ~20% of all invasion events. In contrast, adf cKD, act1 cKO or myoA KO only show an overall invasion rate of ~20% compared to controls. In these cases junctional F-actin accumulation could never be detected. Note that the number of total invasion events for adf cKD, act1cKO and myoAKO correlates to the number of invasion events, seen for RH without F-actin ring formation. Data information: Error bars represent standard deviation. One-way ANOVA analysis was performed for each graph ****p value <0.0001.


F . of posterior F - actin accumulation during invasion of indicated lines . One - way ANOVA was performed for each graph * * * * p value < 0 . 0001 .

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F. Quantification of posterior F-actin accumulation during invasion of indicated parasite lines. One-way ANOVA analysis was performed for each graph ****p value <0.0001.


A . Time - lapse of invading RH Cb - EmeraldFP . During penetration , an F - actin ring is formed at TJ ( orange arrow ) . nucleus ( purple ) is squeezed through TJ ( red arrowhead ) posterior F - actin appears to be directly connected to nucleus TJ ( blue arrow ) . B . of nucleus deformation during live imaging shown in A . nucleus constricts while its passing through TJ ( red arrowheads ) . C . posterior end of deforms during invasion process . F - actin is accumulated during invasion , with posterior end contracting ( blue arrow ) . Data information : Numbers indicate minutes : seconds . Scale bar represents 5 µm .

A. Time-lapse analysis of invading RH Cb-EmeraldFP parasites. During penetration, an F-actin ring is formed at the TJ (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) and posterior F-actin appears to be directly connected to the nucleus and TJ (blue arrow). B. Analysis of nucleus deformation during live imaging shown in A. The nucleus constricts while its passing through the TJ (red arrowheads). C. The posterior end of the parasite deforms during the invasion process. F-actin is accumulated during invasion, with the posterior end contracting (blue arrow). Data information: Numbers indicate minutes:seconds. Scale bar represents 5 µm.


D . Time - lapse stills depicting RH Cb - EmeraldFP invading in absence of detectable F - actin at junction ( orange arrow ) . nucleus ( purple ) is squeezed through TJ ( red arrowhead ) during invasion . Actin accumulation at posterior pole of is strongly detected in cases ( blue arrow ) . E . of nucleus deformation during live imaging shown in ( D ) . nucleus appears to not show constriction once its passing through TJ when F - actin ring is not present . F . posterior end of also deforms during invasion process .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time-lapse stills depicting RH Cb-EmeraldFP parasites invading in absence of detectable F-actin at the junction (orange arrow). The nucleus (purple) is squeezed through the TJ (red arrowhead) during invasion. Actin accumulation at the posterior pole of the parasite is strongly detected in all cases (blue arrow). E. Analysis of nucleus deformation during live imaging shown in (D). The nucleus appears to not show constriction once its passing through the TJ when the F-actin ring is not present. F. The posterior end of the parasite also deforms during the invasion process.


G . Z - stack gallery depicting mid - invading myoA KO . Note that nucleus is located located at back during invasion events . Data information : Scale bar represents 5 µm . arrow points to direction of invasion .

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G. Z-stack gallery depicting mid-invading myoA KO parasites. Note that the nucleus is located located at the back during invasion events. Data information: Scale bar represents 5 µm. White arrow points to direction of invasion.


A . SR - SIM depicting invading with without presence of F - actin ring at tight junction invading adf cKD Cb - EmeraldFP . SAG1 ( in red ) was performed prior to permeabilisation to specifically label extracellular part of . Note accumulation of actin ( blue arrow ) around nucleus posterior pole of , irrespective of F - actin ring formation at TJ ( orange arrowhead ) . 3D rendering is shown for each presented case ( inlet ) . Data information : Scale bar represents 5 µm for SR - SIM arrow points to direction of invasion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SR-SIM images depicting invading parasites with and without the presence of the F-actin ring at the tight junction and invading adf cKD Cb-EmeraldFP parasites. SAG1 staining (in red) was performed prior to permeabilisation to specifically label extracellular part of the parasite. Note the accumulation of actin (blue arrow) around the nucleus and the posterior pole of the parasite, irrespective of F-actin ring formation at the TJ (orange arrowhead). 3D rendering is shown for each presented case (inlet). Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


B . SR image of an invading before entry . F - actin forms a continuous meshwork around nucleus that appears connected to posterior pole . C . F - actin localisation in relation to TJ during invasion . F - actin ( arrow ) is in close vicinity to Ron2 ring ( blue arrow ) during invasion . Data information : cale bar represents 5 µm for SR - SIM hite arrow points to direction of invasion .

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B. SR image of an invading parasite before nuclear entry. F-actin forms a continuous meshwork around the nucleus that appears connected to the posterior pole. C. F-actin localisation in relation to the TJ during invasion. F-actin (white arrow) is in close vicinity to the Ron2 ring (light blue arrow) during invasion. Data information: cale bar represents 5 µm for SR-SIM images hite arrow points to direction of invasion.


D . of perinuclear actin in indicated . were counted by assessing Cb EmeraldFP signal as an actin marker around or in vicinity of nucleus . Still to right represent what was considered as F - actin - nucleus association . orange arrowhead points to F - actin surrounding nucleus in posterior end of , red yellow arrowhead shows F - actin in vicinity of nucleus , hinting at a close interaction between two . Three biological replicates were analysed with a minimum of 25 per replicate . Data information : Error bars represent standard deviation . Scale bar represents 5 µm for SR - SIM arrow points to direction of invasion .

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D. Quantification of perinuclear actin in indicated parasites. Parasites were counted by assessing Cb EmeraldFP signal as an actin marker around or in the vicinity of the nucleus. Still images to the right represent what was considered as F-actin - nucleus association. The orange arrowhead points to F-actin surrounding the nucleus in the posterior end of the parasite, the red and yellow arrowhead shows F-actin in the vicinity of the nucleus, hinting at a close interaction between the two. Three biological replicates were analysed with a minimum of 25 parasites per replicate. Data information: Error bars represent standard deviation. Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


E - G . SR - SIM showing three stages of invading wt . SiR - tubulin for microtubules ( in red ) was performed prior to fixation to specifically label microtubules in . During invasion , microtubules ( MT ) are deformed at TJ ( arrowhead ) . Accumulation of actin ( green arrow ) at posterior forms a F - actin meshwork . nucleus is deformed when it enters TJ ( orange arrowhead ) . H - J . In case of myoA KO , MTs also deform when passing through TJ ( arrowhead ) . However , F - actin is not concentrated at posterior pole or TJ , but evenly distributed within cytosol of , with some peripheral location . K - M . In case of adfKD , MTs are deformed at TJ ( arrowhead ) . F - actin is accumulated at both ends of ( green arrowheads ) , with no accumulation at TJ . Data information : Scale bar represents 5 µm for SR - SIM arrow points to direction of invasion .

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E-G. SR-SIM images showing three stages of invading wt parasites. SiR-tubulin staining for microtubules (in red) was performed prior to fixation to specifically label microtubules in the parasite. During invasion, the microtubules (MT) are deformed at the TJ area (white arrowhead). Accumulation of actin (green arrow) at the posterior forms a F-actin meshwork. The nucleus is deformed when it enters the TJ area (orange arrowhead). H-J. In the case of the myoA KO strain, the MTs also deform when passing through the TJ (white arrowhead). However, F-actin is not concentrated at the posterior pole or the TJ, but evenly distributed within the cytosol of the parasite, with some peripheral location. K-M. In case of adfKD, MTs are deformed at the TJ area (white arrowhead). F-actin is accumulated at both ends of the parasite (green arrowheads), with no accumulation at the TJ. Data information: Scale bar represents 5 µm for SR-SIM images White arrow points to direction of invasion.


A . Different invasion stages of P . falciparum merozoites expressing Cb - EmeraldFP . DAPI labels nucleus ( ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) anti - RON4 labels junction ( red ) . Left panel : onset of invasion . Middle panel : A merozoite in middle of invasion ; note constriction of nucleus ( blue ) as it passes through RON4 junction F - actin colocalising with it ( green ) . Right panel : A merozoite completing invasion ; note F - actin ring beyond RON4 - junction actin filaments surrounding nucleus . Data information : Scale bar represents 1 µm for Plasmodium

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A. Different invasion stages of P. falciparum merozoites expressing Cb-EmeraldFP. DAPI labels the nucleus (DNA), Cb-EmeraldFP labels actin filaments (CB-EME, green) and anti-RON4 labels the junction (red). Left panel: onset of invasion. Middle panel: A merozoite in the middle of invasion; note the constriction of the nucleus (blue) as it passes through the RON4 junction and F-actin colocalising with it (green). Right panel: A merozoite completing invasion; note the F-actin ring beyond the RON4-junction and actin filaments surrounding the nucleus. Data information: Scale bar represents 1 µm for Plasmodium falciparum


B . IFA showing various stages of invasion of erythrocytes by P . knowlesi merozoites . DAPI labels nucleus ( ) , anti - actin Antibody labels actin filaments ( anti - actin , green ) invasion junction is marked with a C - terminally tagged PkRON2 mCherryHA ( red ) . Left panel : a merozoite beginning invasion ; note formation of an F - actin ring ( green ) behind PkRON2 junction ( red ) . Middle panel : A merozoite in middle of invasion event ; note constriction of nucleus ( blue ) as it passes through PkRON2 junction F - actin colocalising with it ( green ) . Right panel : A merozoite completing process of invasion ; note F - actin ring beyond PkRON2 - junction actin filaments colocalising with nucleus . Data information : Scale bar represents 3 µm for Plasmodium knowlesi .

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B. IFA showing various stages of invasion of erythrocytes by P. knowlesi merozoites. DAPI labels the nucleus (DNA), anti-actin Antibody labels actin filaments (anti-actin, green) and the invasion junction is marked with a C-terminally tagged PkRON2 mCherryHA (red). Left panel: a merozoite beginning invasion; note the formation of an F-actin ring (green) behind the PkRON2 junction (red). Middle panel: A merozoite in the middle of the invasion event; note the constriction of the nucleus (blue) as it passes through the PkRON2 junction and F-actin colocalising with it (green). Right panel: A merozoite completing the process of invasion; note the F-actin ring beyond the PkRON2-junction and actin filaments colocalising with the nucleus. Data information: Scale bar represents 3 µm for Plasmodium knowlesi.


C . Super - resolution showing various stages of invasion of erythrocytes by P . falciparum merozoites expressing Cb - EmeraldFP . Left panel : DAPI labels nucleus ( blue ) , Cb - EmeraldFP labels actin filaments ( CB - EME , green ) invasion junction is marked with an anti - RON4 antibody ( red ) . Brightfield have been marked with red dots depicting junction , black dots depicting merozoite boundary still outside erythrocyte dots depicting a merozoite that has penetrated host . Top panel shows an attached merozoite beginning process of invasion , second panel during process of invasion bottom two panels show merozoites completing process of invasion . Right panel : Schematic depicting merozoite invasion into red blood . Letters depict corresponding event in model ( left ) microscopy pictures ( right ) . arrows depict actin filaments that colocalise with nucleus . D . of Plasmodium falciparum invasion events categorising actin - TJ actin - nucleus association events . 121 events were counted in total . Data information : Error bars represent standard deviation . Scale bar represents 1 µm for Plasmodium

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C. Super-resolution images showing various stages of invasion of erythrocytes by P. falciparum merozoites expressing Cb-EmeraldFP. Left panel: DAPI labels the nucleus (blue), Cb-EmeraldFP labels actin filaments (CB-EME, green) and the invasion junction is marked with an anti-RON4 antibody (red). Brightfield images have been marked with red dots depicting the junction, black dots depicting merozoite boundary still outside the erythrocyte and white dots depicting a merozoite that has penetrated the host cell. Top panel shows an attached merozoite beginning the process of invasion, the second panel during the process of invasion and the bottom two panels show merozoites completing the process of invasion. Right panel: Schematic depicting merozoite invasion into red blood cells. Letters depict the corresponding event in the model (left) and microscopy pictures (right). White arrows depict actin filaments that colocalise with the nucleus. D. Quantification of Plasmodium falciparum invasion events categorising actin-TJ and actin-nucleus association events. 121 events were counted in total. Data information: Error bars represent standard deviation. Scale bar represents 1 µm for Plasmodium falciparum


( A ) MYB knockdown ( MiaPaCa - shMYB Panc1 - shMYB ) forced MYB - overexpressing ( BxPC3 - MYB ) PC along with their respective control lines ( MiaPaCa - Scr , Panc1 - Scr , BxPC3 - Neo ) were subjected to hypoxia ( 1 % O2 ) treatment for different time intervals ( 0 - 96 h ) . Their growth was measured by viable counting using a trypan blue exclusion .

(A) MYB knockdown (MiaPaCa-shMYB and Panc1-shMYB) and forced MYB-overexpressing (BxPC3-MYB) PC cells along with their respective control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were subjected to hypoxia (1% O2) treatment for different time intervals (0-96 h). Their growth was measured by viable cell counting using a trypan blue exclusion assay.


( E , F ) ECAR ( E ) OCR ( F ) in control MYB silenced MiaPaCa cultured under normoxia or treated with hypoxia mimetic CoCl2 ( 100 μM for 4 h ) . Thereafter , ECAR was measured in PC with sequential injection of glucose , oligomycin , 2 - DG , while OCR was measured with serial addition of oligomycin , FCCP , antimycin A / rotenone .

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(E, F) ECAR (E) and OCR (F) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 (100 μM for 4 h). Thereafter, ECAR was measured in PC cells with sequential injection of glucose, oligomycin, and 2-DG, while OCR was measured with the serial addition of oligomycin, FCCP, and antimycin A/rotenone.


( A ) MYB expression was analyzed by immunoblotting in pancreatic cancer cultured under hypoxia for different time intervals . HIF1α accumulation was examined as a positive control .

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(A) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals. HIF1α accumulation was examined as a positive control.


MYB ( C ) were measured in prostate ( LNCaP ) , lung ( A549 ) , breast ( MB231 ) , colon ( HCT116 ) cancer cultured under normoxia hypoxia ( 6 h ) by immunoblotting respectively . β - actin served as an internal control . N - normoxia H - hypoxia .

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MYB protein (C) levels were measured in the prostate (LNCaP), lung (A549), breast (MB231), and colon (HCT116) cancer cells cultured under normoxia and hypoxia (6 h) by immunoblotting respectively. β-actin served as an internal control. N- normoxia and H- hypoxia.


( E ) Pancreatic cancer were transiently transfected either with non - targeted control siRNA ( siCtrl ) or HIF1α - targeting siRNA ( siHIF1α ) for 24 h then incubated under hypoxia for 6 h . MYB expression was analyzed at mRNA .

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(E) Pancreatic cancer cells were transiently transfected either with non-targeted control siRNA (siCtrl) or HIF1α -targeting siRNA (siHIF1α) for 24 h and then incubated under hypoxia for 6 h. MYB expression was analyzed at protein and mRNA levels.


( F ) were treated with either vehicle or CoCl2 ( 100µM ) for 6 h , total were isolated . expression of HIF1α MYB was examined . β - actin was used as an internal control .

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(F) Cells were treated with either vehicle or CoCl2 (100µM) for 6 h, and total protein and RNA were isolated. The expression of HIF1α and MYB was examined. β-actin was used as an internal control.


( B ) HuR localization was examined by immunofluorescence microscopy in exposed to hypoxia for 1 h . HuR ( green ) , nucleus ( blue ) . Scale bar , 25 μm ( lower left corner ) . Lower panels are digitally magnified views of marked rectangles .

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(B) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h. HuR (green), and nucleus (blue). Scale bar, 25 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles.


( C ) fractions were prepared from cultured under hypoxia for 1h HuR expression was examined by immunoblotting . α - Tubulin ( ) Lamin A ( cytoplasm ) were used as loading controls .

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(C) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting. α-Tubulin (nuclear) and Lamin A (cytoplasm) were used as loading controls.


( G ) transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h , MYB HuR expression was determined by immunoblotting or qRT - PCR .

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(G) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h, and MYB and HuR expression was determined by immunoblotting or qRT-PCR.


( A ) MYB - silenced ( MiaPaCa - shMYB Panc1 - shMYB ) MYB - overexpressing ( BxPC3 - MYB ) PC , along with their control lines ( MiaPaCa - Scr , Panc1 - Scr , BxPC3 - Neo ) were exposed to hypoxia for 6 h . lysates were made , expression of MYB HIF1α was examined by immunoblotting . β - actin served as a loading control .

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(A) MYB-silenced (MiaPaCa-shMYB and Panc1-shMYB) and MYB-overexpressing (BxPC3-MYB) PC cells, along with their control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were exposed to hypoxia for 6 h. Protein lysates were made, and the expression of MYB and HIF1α was examined by immunoblotting. β-actin served as a loading control.


( C ) transcriptional activity of HIF1α promoter was determined by performing promoter - reporter . MYB - modulated PC lines were transfected with reporter plasmid , after 24 h of transfection , were cultured under hypoxia or normoxia for 24 h , supernatant was collected . HIF1α promoter - driven Gaussia luciferase activity was normalized with SEAP activity , data was presented as a bar diagram .

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(C) The transcriptional activity of the HIF1α promoter was determined by performing the promoter-reporter assay. MYB-modulated PC cell lines were transfected with the reporter plasmid, and after 24 h of transfection, cells were cultured under hypoxia or normoxia for 24 h, and the supernatant was collected. HIF1α promoter-driven Gaussia luciferase activity was normalized with SEAP activity, and the data was presented as a bar diagram.


( E ) MYB - silenced or - overexpressing PC lines were exposed to normoxia or hypoxia for 6 h subjected to immunoprecipitation using anti - MYB antibodies or IgG control followed by qPCR using site - specific primers .

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(E) MYB-silenced or -overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti-MYB antibodies or IgG control followed by qPCR analysis using site-specific primers.


( A ) Immunoblot was performed to analyze HIF1α MYB expression in MYB - silenced ( shMYB ) those having a forced overexpression of HIF1α ( shMYB - HIF1αOE ) along with their respective controls ( Scr shMYB - Neo ) cultured under hypoxia . β - actin served as a loading control .

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(A) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB-silenced (shMYB) and those having a forced overexpression of HIF1α (shMYB-HIF1αOE) along with their respective controls (Scr and shMYB-Neo) cultured under hypoxia. β-actin served as a loading control.


( C ) Live / Dead was performed in MYB - silenced MiaPaCa Panc - 1 or MYB silenced - HIF1α overexpressed cultured under hypoxia for 48 h . were visualized under a microscope . Representative are presented . number of live dead was counted from captured random . Scale bar , 150 μm ( lower right corner ) .

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(C) Live/Dead staining was performed in MYB-silenced MiaPaCa and Panc-1 or MYB silenced- HIF1α overexpressed cells cultured under hypoxia for 48 h. Cells were visualized under a fluorescence microscope. Representative fluorescence micrographs are presented. The number of live and dead cells was counted from the captured random images. Scale bar, 150 μm (lower right corner).


( F ) MYB - silenced HIF1α overexpressing MYB knockdown PC control were cultured under hypoxia for 48 h . Conditioned media was collected analyzed to assess glucose consumption lactate secretion by measuring their . data was normalized with number of presented as relative fold changes .

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(F) MYB-silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h. Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels. The data was normalized with the number of cells and presented as relative fold changes.


( C ) Quantitative ChIP - qPCR was performed to analyze occupancy of MYB under normoxia hypoxia at promoter regions of selected glycolytic genes ( GLUT3 , HK2 , PFKL , ENO2 , MCT4 ) using anti - MYB or IgG control antibodies . enrichment of promoter regions was compared between cultured under normoxia or hypoxia .

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(C) Quantitative ChIP-qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes (GLUT3, HK2, PFKL, ENO2, and MCT4) using anti-MYB or IgG control antibodies. The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia.


p300 ( E ) , H3K9ac ( F ) on promoter regions were also confirmed by ChIP - qPCR using specific antibodies fold enrichment calculated presented as a bar graph after normalization with control IgG .

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p300 (E), and H3K9ac (F) on the promoter regions were also confirmed by ChIP- qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG.


( A , B ) Interaction of MYB with HIF1α ( A ) p300 ( B ) was examined by co - immunoprecipitation assays in lysates of pancreatic cancer cultured under hypoxia for 6 h . Immunoprecipitation with IgG isotype antibody was used as a control . 10 % of total lysate was used as input for western blot .

(A, B) Interaction of MYB with HIF1α (A) and p300 (B) was examined by co-immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h. Immunoprecipitation with IgG isotype antibody was used as a control. 10% of total cell lysate was used as input for western blot analysis.


( C , D ) Co - localization of MYB with HIF1α ( C ) p300 ( D ) was examined in pancreatic cancer grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy . Scale bar , 30 μm ( lower left corner ) . Lower panels are digitally magnified views of marked rectangles . For ease of visualization , a pseudo color ( purple ) was adopted for p300 .

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(C, D) Co-localization of MYB with HIF1α (C) and p300 (D) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy. Scale bar, 30 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles. For ease of visualization, a pseudo color (purple) was adopted for p300 staining.


( B , C ) growth was monitored by non - invasive bioluminescence imaging . Tumors were resected at experimental endpoint , their weights ( B ) volumes ( C ) were recorded .

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(B, C) Tumor growth was monitored by non-invasive bioluminescence imaging. Tumors were resected at the experimental endpoint, and their weights (B) and volumes (C) were recorded.


( G ) xenograft serial sections were immunostained for pimonidazole ( PDZ ) adduct , MYB , HK2 , GLUT3 , MCT4 using specific antibodies . were taken at 40X magnification . Scale bar , 60 μm ( lower left corner ) . dashed lines demarcate hypoxic region on PDZ pattern .

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(G) Tumor xenograft serial sections were immunostained for pimonidazole (PDZ) adduct, MYB, HK2, GLUT3, and MCT4 using specific antibodies. Images were taken at 40X magnification. Scale bar, 60 μm (lower left corner). The dashed lines demarcate the hypoxic region based on the PDZ staining pattern.


( A - C ) AFM of unloaded ( u - NPs ) cholesterol loaded NPs ( NPs - Chol ) . AFM " height " ( left column ) , 3D reconstruction ( middle column ) micrograph ( right column ) of u - NPs ( A ) , NPs - Chol1 ( B ) NPs - Chol3 ( C ) .

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(A-C) AFM and TEM analysis of unloaded (u-NPs) and cholesterol loaded NPs (NPs-Chol). AFM "height" images (left column), 3D reconstruction (middle column) and TEM micrograph (right column) of u-NPs (A), NPs-Chol1 (B) and NPs-Chol3 (C).


( D ) Release profile in water of cholesterol ( continuous , - ) NBD - Chol ( dotted , - - - ) from NPs - Chol1 NPs - NBD - Chol1 , respectively .

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(D) Release profile in water of cholesterol (continuous line, -) and NBD-Chol (dotted line, - - -) from NPs-Chol1 and NPs-NBD-Chol1, respectively.


( E ) In vitro release of NBD - Chol from NPs at different time - intervals in NS . Data in graph represent mean ( µg ) ± of total NBD - Chol ( embedded into released from NPs ; red columns ) NBD - Chol released after NPs degradation ( purple columns ) present in homogenates of NS treated with NPs - NBD -

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(E) In vitro release of NBD-Chol from NPs at different time-intervals in NS cells. Data in the graph represent mean (µg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1


( A - B ) Representative confocal of liver ( A ) brain ( B ) slices from R6 / 2 mice ip injected with C - NPs ( left ) or with g7 - NPs ( right ) sacrificed after 4 hrs . ( C ) of g7 - NPs localized in liver , striatum , cortex of WT ( n = 3 ) R6 / 2 mice ( n = 3 ) . Data represent mean ( µg ) ± .

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(A-B) Representative confocal images of liver (A) and brain (B) slices from R6/2 mice ip injected with C-NPs (left) or with g7-NPs (right) and sacrificed after 4 hrs. (C) Quantification of g7-NPs localized in the liver, striatum, and cortex of WT (n=3) and R6/2 mice (n=3). Data represent mean (µg) ± SEM.


( D - E ) g7 - NPs in brain slices from R6 / 2 mice administered with a single ip injection sacrificed after 24 hrs ( D , left ) or 2 ( D , right ) after multiple ip injections within 1 week ( E ) .

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(D-E) g7-NPs in brain slices from R6/2 mice administered with a single ip injection and sacrificed after 24 hrs (D, left) or 2 weeks (D, right) and after multiple ip injections within 1 week (E).


( F - I ) Representative confocal of immunostaining against IBA1 ( F ) , GFAP ( G ) , calbindin ( H ) , DARPP - 32 ( I ) on coronal sections of brains isolated from R6 / 2 mice ip injected with g7 - NPs sacrificed at indicated time points . arrowheads indicate intracellular g7 - NPs .

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(F-I) Representative confocal images of immunostaining against IBA1 (F), GFAP (G), calbindin (H), and DARPP-32 (I) on coronal sections of brains isolated from R6/2 mice ip injected with g7-NPs and sacrificed at the indicated time points. White arrowheads indicate intracellular g7-NPs.


( A ) Representative confocal image ( crop ) of brain slices from R6 / 2 mice ip injected with rhodamine labeled g7 - NPs - NBD - Chol sacrificed after 12 hrs , showing co - localization of NBD - Chol ( green ) Rhodamine ( NPs , red ) . Scale bar : 5m .

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(A) Representative confocal image (crop) of brain slices from R6/2 mice ip injected with rhodamine labeled g7-NPs-NBD-Chol and sacrificed after 12 hrs, showing co-localization of NBD-Chol (green) and Rhodamine (NPs, red). Scale bar: 5m.


( B - C ) Representative confocal image ( low magnification ) of brain slices from R6 / 2 mice ip injected with g7 - NPs - NBD - Chol sacrificed after 24 hrs ( B ) or 2 ( C ) relative co - localization of NBD - Chol g7 - NPs . Scale bar : 10 m ( D ) g7 - NPs in brain slices at same time points in ( B - C ) . DAPI was used to counterstain nuclei . Data in ( D ) are expressed as number of g7 - NPs ( evaluated their ) for 100 m2 . Statistics : * * P < 0 , 01 ( 48 hrs vs 7 days ; 7 days vs 14 days ) , * * * P < 0 , 001 ( 24 hrs vs 7 days ; 7 days vs 14 days ) determined by one - way ANOVA followed by Newmann - Keuls multiple comparison .

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(B-C) Representative confocal image (low magnification) of brain slices from R6/2 mice ip injected with g7-NPs-NBD-Chol and sacrificed after 24 hrs (B) or 2 weeks (C) and relative co-localization of NBD-Chol and g7-NPs. Scale bar: 10 m (D) g7-NPs quantification in brain slices at the same time points in (B-C). DAPI was used to counterstain nuclei. Data in (D) are expressed as number of g7-NPs (evaluated based their size) for 100 m2 sem. Statistics: **P<0,01 (48 hrs vs 7 days; 7 days vs 14 days), ***P<0,001 (24 hrs vs 7 days; 7 days vs 14 days) determined by one-way ANOVA followed by Newmann-Keuls multiple comparison test.


( A , D ) Spontaneous IPSCs ( A ) spontaneous EPSCs ( D ) were recorded from striatal MSNs ( WTs = 52 ; R6 / 2 - untreated = 27 ; R6 / 2 - Chol = 29 ) at a holding potential of + 10 mV - 70mV respectively . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled . ( B , E ) Amplitude - frequency histogram average frequency ( inset ) of IPSCs ( B ) EPSCs ( E ) from R6 / 2 - Chol , R6 / 2 - untreated WT MSNs . ( C , F ) Cumulative inter - event histogram showing release probability of IPSCs ( C ) EPSCs ( F ) in groups .

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(A,D) Spontaneous IPSCs (A) and spontaneous EPSCs (D) were recorded from striatal MSNs (WTs=52; R6/2-untreated=27; R6/2-Chol=29) at a holding potential of +10 mV and -70mV respectively. As no differences were found between R6/2 mice treated with saline (R6/2) or with empty g7-NPs (R6/2-emp), data were pooled. (B, E) Amplitude-frequency histogram and average frequency (inset) of IPSCs (B) and EPSCs (E) from R6/2-Chol, R6/2-untreated and WT MSNs. (C,F) Cumulative inter-event histogram showing the release probability of IPSCs (C) and EPSCs (F) in all groups.


( B ) Fall latency from an accelerating rotarod for 5 - 11 - week - old WT R6 / 2 mice during cholesterol supplementation ( WT = 17 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 13 ) .

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(B) Fall latency from an accelerating rotarod for 5-11-week-old WT and R6/2 mice during cholesterol supplementation (WT=17; R6/2-untreated=21; R6/2-Chol=13).


( C ) Rearing activity in open field at 10 of ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

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(C) Rearing activity in open field at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( D ) global activity in open field at 10 of ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

(D) global activity in the open field test at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( E ) Index of discrimination ( % ) in WT , R6 / 2 - untreated R6 / 2 - Chol mice during disease progression , at 8 of ( WT = 24 ; R6 / 2 - untreated = 36 ; R6 / 2 - Chol = 21 ) , at 10 of ( WT = 25 ; R6 / 2 - untreated = 35 ; R6 / 2 - Chol = 20 ) at 12 of ( WT = 24 ; R6 / 2 - untreated = 30 ; R6 / 2 - Chol = 19 ) ; index above zero indicates a preference for novel object ; index below zero indicates a preference for familiar object . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or treated with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled .

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(E) Index of discrimination (%) in WT, R6/2-untreated and R6/2-Chol mice during disease progression, at 8 weeks of age (WT=24; R6/2-untreated=36; R6/2-Chol=21), at 10 weeks of age (WT=25; R6/2-untreated=35; R6/2-Chol=20) and at 12 weeks of age (WT=24; R6/2-untreated=30; R6/2-Chol=19); the index above zero indicates a preference for the novel object; the index below zero indicates a preference for the familiar object. As no differences were found between R6/2 mice treated with saline (R6/2) or treated with empty g7-NPs (R6/2-emp), data were pooled.


( A - B ) ( A ) relative densitometry ( B ) of several synaptic in triton - insoluble ( synaptic enriched ) fractions purified from total brains . of PSD95 NMDA receptor subunits GluN1 GluN2B are rescued in R6 / 2 mice by cholesterol supplementation .

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(A-B) protein levels (A) and relative densitometry quantification (B) of several synaptic proteins in triton-insoluble (synaptic enriched) fractions purified from total brains. Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6/2 mice by cholesterol supplementation.


( C - D - E ) mRNA for Bdnf ( C ) , Snap25 ( D ) in cortex hippocampus ; Complexin II ( E ) in cortex , hippocampus striatum from a subset of WT ( n = 4 ) , R6 / 2 - untreated ( n = 7 ) R6 / 2 - chol ( n = 3 ) . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D-E) mRNA levels for Bdnf (C), Snap25 (D) in cortex and hippocampus; Complexin II (E) in cortex, hippocampus and striatum from a subset of WT (n=4), R6/2-untreated (n=7) and R6/2-chol animals (n=3). As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( F - G ) Representative of Nissl ( F ) ventricle volume revealed by Neurolucida at 12 of in WT ( n = 7 ) , R6 / 2 ( n = 7 ) , R6 / 2 - emp ( n = 6 ) , R6 / 2 - Chol ( n = 8 )

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(F-G) Representative images of Nissl Staining (F) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT (n= 7), R6/2 (n=7), R6/2-emp (n=6), R6/2-Chol (n=8)


( A - B ) Lathosterol 24OHC measured by mass spectrometry in brain of WT ( n = 6 ) , R6 / 2 - untreated ( n = 7 ) R6 / 2 - Chol ( n = 5 ) mice . Data represent mean ± .

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(A-B) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT (n=6), R6/2-untreated (n=7) and R6/2-Chol (n=5) mice. Data represent mean ± SEM.


( C - D ) mRNA of hmgcr fdft1 in liver lung of WT ( n = 7 ) , R6 / 2 - untreated ( n = 8 ) R6 / 2 - Chol ( n = 4 ) mice . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D) mRNA levels of hmgcr and fdft1 in liver and lung of WT (n=7), R6/2-untreated (n=8) and R6/2-Chol (n=4) mice. As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( E - F ) mRNA of inflammatory genes in liver lung of of WT ( n = 7 ) , R6 / 2 ( n = 4 ) , R6 / 2 - emp ( n = 4 ) R6 / 2 - Chol ( n = 5 ) mice .

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(E-F) mRNA levels of inflammatory genes in liver and lung of the of WT (n=7), R6/2 (n=4), R6/2-emp (n=4) and R6/2-Chol (n=5) mice.


A Immunoblot of knockdown of exogenous USP19 in 293T expressing Flag - USP19 ( top ) or endogenous USP19 in HeLa ( bottom ) treated with USP19 - specific siRNA or scrambled ( Scr ) siRNA .

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A Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag-USP19 (top) or endogenous USP19 in HeLa cells (bottom) treated with USP19-specific siRNA or scrambled (Scr) siRNA.


B Representative of GFP - LC3B in HeLa - GFP - LC3B transfected with USP19 - specific siRNA or scrambled siRNA during growth in normal medium ( DMEM ) or EBSS medium for 3 h . Arrows denote representative autophagosomes . Scale bar , 200 μm .

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B Representative images of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA during growth in normal medium (DMEM) or EBSS medium for 3 h. Arrows denote representative autophagosomes. Scale bar, 200 μm.


C of GFP - LC3B in HeLa - GFP - LC3B transfected with USP19 - specific siRNA or scrambled siRNA . Bars represented mean ± standard error of mean ( ) of triplicate samples ( 20 per sample ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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C Quantification of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA. Bars represented mean ± standard error of the mean (SEM) of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.


D Immunoblot detection of relative accumulation of LC3 I LC3 II in HeLa transfected with USP19 - specific siRNA or scrambled siRNA with rapamycin ( 250 nM ) treatment for 12 h .

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D Immunoblot detection of the relative accumulation of LC3 I and LC3 II in HeLa cells transfected with USP19-specific siRNA or scrambled siRNA with rapamycin (250 nM) treatment for 12 h.


E HeLa were transfected with USP19 - specific siRNA or scrambled siRNA treated with indicated of rapamycin for 12 h , p62 were detected by immunoblot .

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E HeLa cells were transfected with USP19-specific siRNA or scrambled siRNA and treated with indicated concentration of rapamycin for 12 h, p62 levels were detected by immunoblot.


F Human peripheral blood mononuclear ( PBMCs ) transfected with control or USP19 - specific siRNA were treated with rapamycin ( 250 nM ) for 18 h , lysates were analyzed with each antibody .

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F Human peripheral blood mononuclear cells (PBMCs) transfected with control or USP19-specific siRNA were treated with rapamycin (250 nM) for 18 h, the lysates were analyzed with each antibody.


G Flag - USP19 stably expressing 293T were incubated with DMEM or EBSS medium for 3 h in absence or presence of 50 μM of chloroquine ( CQ ) . were then assayed for relative accumulation of LC3 - I LC3 - II by immunoblot using anti - LC3 antibody .

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G Flag-USP19 stably expressing 293T cells were incubated with DMEM or EBSS medium for 3 h in the absence or presence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblot analysis using anti-LC3 antibody.


H Flag - USP19 inducible HeLa were treated with 200 ng / ml doxycycline ( Doxy ) for overnight to induce expression of Flag - USP19 . of p62 were analyzed after rapamycin treatment for 12 h at indicated concentrations .

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H Flag-USP19 inducible HeLa cells were treated with 200 ng/ml doxycycline (Doxy) for overnight to induce the expression of Flag-USP19. The protein levels of p62 were analyzed after rapamycin treatment for 12 h at indicated concentrations.


I GFP - LC3B stably expressing HeLa were transfected with pcDNA3 . 1 empty vector or plasmid expressing wild - type mutant forms of USP19 ( CS or CS / HA ) . Pictures in ( B ) ( I ) were taken using Leica DMI3000 B microscope with a × 100 oil - immersion objective . Scale bar , 200 μm . J Average GFP - LC3B per were calculated . Bars represented mean ± of triplicate samples ( 20 per sample ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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I GFP-LC3B stably expressing HeLa cells were transfected with pcDNA3.1 empty vector or plasmid expressing the wild-type and the mutant forms of USP19 (CS or CS/HA). Pictures in (B) and (I) were taken using Leica DMI3000 B microscope with a×100 oil-immersion objective. Scale bar, 200 μm.J Average GFP-LC3B puncta per cell were calculated. Bars represented mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.


A 293T were transfected with plasmids encoding HA - USP19 Flag - tagged Beclin - 1 members ( Flag - ATG14L , Flag - VPS15 , Flag - Beclin - 1 , Flag - VPS34 , Flag - UVRAG Flag - AMBRA1 ) , followed by immunoprecipitation ( IP ) with anti - Flag beads immunoblot with anti - HA . Throughout was immunoblot of whole - lysates ( WCL ) without immunoprecipitation .

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A 293T cells were transfected with plasmids encoding HA-USP19 and Flag-tagged Beclin-1 complex members (Flag-ATG14L, Flag-VPS15, Flag-Beclin-1, Flag-VPS34, Flag-UVRAG and Flag-AMBRA1), followed by immunoprecipitation (IP) with anti-Flag beads and immunoblot analysis with anti-HA. Throughout was the immunoblot analysis of whole-cell lysates (WCL) without immunoprecipitation.


B , C Extracts of A549 ( B ) or human PBMCs ( C ) incubated with EBSS for various time points ( above lanes ) were subjected to immunoprecipitation with anti - Beclin - 1 immunoblot with indicated antibodies ( shown on left ) .

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B, C Extracts of A549 cells (B) or human PBMCs (C) incubated with EBSS for various time points (above lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with indicated antibodies (shown on the left).


D HeLa were incubated in EBSS medium for 1 h after co - transfection eGFP - USP19 dsRed - Beclin - 1 for 48 h . were imaged for eGFP dsRed . Scale bar , 200 μm . E Quantitative data are mean ± of triplicate samples ( 20 per sample ) . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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D HeLa cells were incubated in EBSS medium for 1 h after co-transfection eGFP-USP19 and dsRed-Beclin-1 for 48 h. Cells were imaged for eGFP and dsRed. Scale bar, 200 μm.E Quantitative data are mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.


F Co - immunoprecipitation immunoblot of 293T transfected with deletion mutants of USP19 plasmid along with vector encoding Flag - Beclin - 1 .

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F Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of USP19 plasmid along with vector encoding Flag-Beclin-1.


G Co - immunoprecipitation immunoblot of 293T transfected with deletion mutants of Beclin - 1 plasmid along with vector encoding HA - USP19 .

G Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of Beclin-1 plasmid along with vector encoding HA-USP19.


A Immunoblot of extracts of 293T transfected with expression vector for Flag - Beclin - 1 increasing doses of expression vector for HA - USP19 ( wedge ) .

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A Immunoblot analysis of extracts of 293T cells transfected with expression vector for Flag-Beclin-1 and increasing doses of expression vector for HA-USP19 (wedge).


B HeLa harboring inducible Flag - USP19 was induced with indicated of Doxy for overnight , was harvested for immunoblot .

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B HeLa cell line harboring inducible Flag-USP19 was induced with indicated concentration of Doxy for overnight, and the protein was harvested for immunoblot analysis.


C BECN1 mRNA of same sample ( B ) were detected by real - time PCR . Bars represented mean ± of triplicate samples .

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C BECN1 mRNA levels of the same sample (B) were detected by real-time PCR. Bars represented mean ± SEM of triplicate samples.


D Human PBMCs were transfected with USP19 - specific or scramble siRNA treated with rapamycin ( 250 nM ) for 18 h . extracts were analyzed by immunoblot with indicated antibodies .

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D Human PBMCs were transfected with USP19-specific or scramble siRNA and treated with rapamycin (250 nM) for 18 h. The extracts were analyzed by immunoblot with indicated antibodies.


E Wild - type ( WT ) USP19 KO A549 were cultured in EBSS for indicated time points , expression of Beclin - 1 ULK1 were detected by immunoblot .

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E Wild-type (WT) and USP19 KO A549 cells were cultured in EBSS for indicated time points, and the protein expression levels of Beclin-1 and ULK1 were detected by immunoblot.


F USP13 KO HEK293T were transfected with plasmids encoding Myc - USP5 , Myc - USP19 Myc - USP38 extracts were analyzed by immunoblot .

F USP13 KO HEK293T cells were transfected with plasmids encoding Myc-USP5, Myc-USP19 and Myc-USP38 and the cellular extracts were analyzed by immunoblot.


G 293T transfected with plasmid expressing Flag - Beclin - 1 empty vector or Myc - tagged USP19 ( WT , CS , CS / HA or ΔTMD ) , lysates were analyzed by immunoblot .

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G 293T cells transfected with plasmid expressing Flag-Beclin-1 and empty vector or Myc-tagged USP19 (WT, CS, CS/HA or ΔTMD), the lysates were analyzed by immunoblot.


H 293T were transfected with plasmid encoding Flag - Beclin - 1 treated with CHX ( 100 μg / ml ) alone or with MG132 ( 10 μM ) , 3MA ( 10 mM ) , CQ ( 50 μM ) as combination for 6 h or transfected with HA - USP19 plasmid . lysates were analyzed by immunoblot .

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H 293T cells were transfected with plasmid encoding Flag-Beclin-1 and treated with CHX (100 μg/ml) alone or with MG132 (10 μM), 3MA (10 mM), CQ (50 μM) as combination for 6 h or transfected with the HA-USP19 plasmid. The cell lysates were analyzed by immunoblot.


A HeLa were transfected with USP19 siRNA or scrambled siRNA extracts were harvested after MG132 ( 10 μM ) treatment for 3 h . extracts were immunoprecipitated using anti - Beclin - 1 antibody or with IgG as a negative control analyzed by immunoblot using anti - ubiquitin anti - Beclin - 1 antibodies .

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A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 (10 μM) treatment for 3 h. Protein extracts were immunoprecipitated using anti-Beclin-1 antibody or with IgG as a negative control and analyzed by immunoblot using anti-ubiquitin and anti-Beclin-1 antibodies.


B Lysates of 293T transfected with plasmid expressing Flag - Beclin - 1 HA - tagged ubiquitin ( HA - Ub ( wild type ) , K6 - linked - Ub , K11 - linked - Ub , K27 - linked - Ub , K29 - linked - Ub , K33 - linked - Ub , K48 - linked - Ub or K63 - linked - Ub ) , together with empty vector or expression vector of Myc - USP19 treated with MG132 ( 10 μM ) for 3 h were immunoprecipitated with anti - Flag immunoblotted with anti - HA .

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B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.


C HeLa were transfected with USP19 - specific siRNA plasmid encoding Flag - Beclin - 1 , plus K11 - linked - Ub . was harvested after MG132 ( 10 μM ) treatment for 3 h immunoprecipitated with anti - Flag immunoblotted with anti - HA .

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C HeLa cells were transfected with USP19-specific siRNA and plasmid encoding Flag-Beclin-1, plus K11-linked-Ub. Protein was harvested after MG132 (10 μM) treatment for 3 h and immunoprecipitated with anti-Flag and immunoblotted with anti-HA.


D 293T were transfected with plasmid for Flag - Beclin - 1 HA - K11 - Ub , together with empty vector or expression vector for Myc - USP19 ( WT , CS or CS / HA ) treated with MG132 ( 10 μM ) . lysates were subjected to immunoprecipitation with anti - Flag immunoblot with anti - HA .

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D 293T cells were transfected with plasmid for Flag-Beclin-1 and HA-K11-Ub, together with the empty vector or expression vector for Myc-USP19 (WT, CS or CS/HA) and treated with MG132 (10 μM). Cell lysates were subjected to immunoprecipitation with anti-Flag and immunoblot analysis with anti-HA.


E Purified ubiquitinated Beclin - 1 was incubated with immunopurified Flag - USP19 in vitro in deubiquitinating buffer . immunoblot was probed with anti - HA .

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E Purified ubiquitinated Beclin-1 was incubated with immunopurified Flag-USP19 in vitro in deubiquitinating buffer. The immunoblot was probed with anti-HA.


A Lysates of 293T transfected with plasmid for Flag - Beclin - 1 HA - K11 - linked - Ub , together with empty vector or expression vector for Myc - USP5 or Myc - USP19 were immunoprecipitated with anti - Flag immunoblotted with anti - HA .

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A Lysates of 293T cells transfected with plasmid for Flag-Beclin-1 and HA-K11-linked-Ub, together with empty vector or expression vector for Myc-USP5 or Myc-USP19 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.


B Immunoprecipitation immunoblot of 293T transfected with vectors expressing Flag - Beclin - 1 HA - K11 - linked ubiquitin .

B Immunoprecipitation and immunoblot analysis of 293T cells transfected with vectors expressing Flag-Beclin-1 and HA-K11-linked ubiquitin.


C 293T were transfected with Flag - Beclin - 1 ( WT or K437R ) treated with CHX ( 100 μg / ml ) for indicated time points . expression of WT K437R Flag - Beclin - 1 were analyzed by immunoblot . D of expression of WT K437R Flag - Beclin - 1 .

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

C 293T cells were transfected with Flag-Beclin-1 (WT or K437R) and treated with CHX (100 μg/ml) for indicated time points. The expression levels of WT and K437R Flag-Beclin-1 were analyzed by immunoblot.D Quantification of the expression levels of WT and K437R Flag-Beclin-1.


E Immunoblot of extracts of 293T transfected with empty vector or vector for HA - USP19 , together with plasmid expressing WT or K427R , K430R or K437R mutant of Flag - Beclin - 1 .

E Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA-USP19, together with plasmid expressing WT or K427R, K430R or K437R mutant of Flag-Beclin-1.


F HeLa transfected with BECN1 sgRNA restored with WT K437R Flag - Beclin - 1 were stained by anti - LC3 antibody . Scale bar , 200 μm . G of LC3 shown in ( F ) . Bars represented mean ± of triplicate samples . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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

F HeLa cells transfected with BECN1 sgRNA and restored with WT and K437R Flag-Beclin-1 were stained by anti-LC3 antibody. Scale bar, 200 μm.G Quantification of LC3 puncta shown in (F). Bars represented mean ± SEM of triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001.


A Luciferase activity in 293T transfected with an ISRE ( left ) or IFN - β ( right ) promoter - driven luciferase reporter together with plasmid encoding RIG - I ( N ) or cGAS STING .

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A Luciferase activity in 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter and together with plasmid encoding RIG-I (N) or cGAS and STING.


B lysates of WT USP19 KO A549 infected with SeV at indicated time points were immunoblotted with indicated antibodies .

B Protein lysates of WT and USP19 KO A549 cells infected with SeV at indicated time points were immunoblotted with indicated antibodies.


C RT - PCR of gene transcription in WT USP19 KO A549 after infection with SeV at indicated time points .

C RT-PCR analysis of gene transcription in WT and USP19 KO A549 cells after infection with SeV at indicated time points.


D Phase - contrast microscopy ( PH ) microscopy of WT USP19 KO A549 infected with VSV - eGFP at an MOI of 0 . 01 for indicated time . Scale bar , 50 μm .

D Phase-contrast microscopy (PH) and fluorescence microscopy images of WT and USP19 KO A549 cells infected with VSV-eGFP at an MOI of 0.01 for the indicated time length. Scale bar, 50 μm.


E Human PBMCs were transfected with control or USP19 - specific siRNA , followed by treatment with influenza A / Puerto Rico / 8 / 34 ( H1N1 ) ( PR8 ) ( MOI = 5 ) at different time points , lysates were analyzed with each antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Human PBMCs were transfected with control or USP19-specific siRNA, followed by treatment with influenza A/Puerto Rico/8/34 (H1N1) (PR8) (MOI = 5) at different time points, the lysates were analyzed with each antibody.


F Human PBMCs were transfected with control or USP19 - specific siRNA were infected with H1N1 ( MOI = 5 ) at different time points . Relative expression of IFNB , ISG15 , ISG54 , ISG56 mRNA H1N1 nucleoprotein ( NP ) were measured by real - time PCR .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Human PBMCs were transfected with control or USP19-specific siRNA were infected with H1N1 virus (MOI = 5) at different time points. Relative expression levels of IFNB, ISG15, ISG54, ISG56 mRNA and H1N1 nucleoprotein (NP) RNA were measured by real-time PCR.


G Relative expression of IFNB mRNA interferon - stimulated genes , including ISG54 ISG56 in WT , BECN1 , ATG5 SQSTM1 KO with or without USP19 .

G Relative expression levels of IFNB mRNA and interferon-stimulated genes, including ISG54 and ISG56 in WT, BECN1, ATG5 and SQSTM1 KO cells with or without USP19.


H , I Luciferase activity in WT BECN1 KO 293T transfected with an ISRE ( H ) or IFN - β ( I ) promoter - driven luciferase reporter after infection with SeV for indicated time points .

H, I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (H) or IFN-β (I) promoter-driven luciferase reporter after infection with SeV for indicated time points.


A Luciferase activity in WT BECN1 KO 293T transfected with an ISRE ( left ) or IFN - β ( right ) promoter - driven luciferase reporter after infection with SeV for 12 h .

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A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter after infection with SeV for 12 h.


B WT BECN1 KO 293T were infected with SeV for indicated time points , then extracts were analyzed by immunoblot using 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

B WT and BECN1 KO 293T cells were infected with SeV for indicated time points, then protein extracts were analyzed by immunoblot using the indicated antibodies.


C extracts of THP - 1 infected with SeV for various time points ( upper lanes ) were subjected to immunoprecipitation with anti - Beclin - 1 immunoblot with antibodies shown on left .

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C Protein extracts of THP-1 cells infected with SeV for various time points (upper lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with antibodies shown on the left.


D Co - immunoprecipitation immunoblot of lysates of A549 infected with SeV .

D Co-immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV.


E 293T were transfected with vectors encoding HA - Beclin - 1 Flag - MAVS or Flag - MAVSΔCARD , followed by IP with anti - Flag beads immunoblot with anti - HA .

E 293T cells were transfected with vectors encoding HA-Beclin-1 and Flag-MAVS or Flag-MAVSΔCARD, followed by IP with anti-Flag beads and immunoblot analysis with anti-HA.


F Co - immunoprecipitation immunoblot of WT BECN1 KO 293T transfected with HA - MAVS plasmid along with vector encoding Flag - RIG - I ( N ) in absence or presence of USP19 .

F Co-immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA-MAVS plasmid along with vector encoding Flag-RIG-I (N) in the absence or presence of USP19.


G extracts of scramble or USP19 - specific siRNA treated 293T transfected with HA - MAVS plasmid along with vector encoding for Flag - RIG - I ( N ) in absence or presence of Beclin - 1 were subjected to immunoprecipitation immunoblot .

O O O O O O O O O O O O O O O O O O O O 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 Protein extracts of scramble or USP19-specific siRNA treated 293T cells transfected with HA-MAVS plasmid along with vector encoding for Flag-RIG-I (N) in the absence or presence of Beclin-1 were subjected to immunoprecipitation and immunoblot analysis.


H Luciferase activity in 293T transfected with scramble or USP19 - specific siRNA ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 .

O O O O O O 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 Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin-1.


I Luciferase activity in 293T transfected with scramble or USP19 - specific siRNA IFN - β luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 .

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

I Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and IFN-β luciferase reporter after SeV infection for indicated time points with or without Beclin-1.


( B ) Violinplots showing number of detected mRNAs lincRNAs across distinct types by 10x Genomics . Total 7 , 935 were collected from 2 replicates of 6 mice .

(B) Violinplots showing the number of detected mRNAs and lincRNAs across distinct cell types by 10x Genomics. Total 7,935 cells were collected from 2 replicates of 6 mice.


( C ) Boxplot showing number of detected lincRNAs across distinct neuron types by two scRNA - seq methods . Total 1 , 818 neurons from 10x Genomics were collected from 2 replicates of 6 mice . 197 neurons from Smart - seq2 ( analyzed in B , C D ) were collected from lumbar 5 DRG from five adult mice as described in previous study .

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(C) Boxplot showing the number of detected lincRNAs across distinct neuron types by two scRNA-seq methods. Total 1,818 neurons from 10x Genomics were collected from 2 replicates of 6 mice. And 197 neurons from Smart-seq2 (analyzed in B, C and D) were collected from lumbar 5 DRG from five adult male mice as described in previous study.


( E ) Representative ( Left ) of smFISH in DRG section , tSNE plot of 10x Genomics ( Middle ) , quantitative result ( Right ) showed that C530044C16Rik was co - localized with Gal - Cre ; Ai9 tdTomato neurons ( n = 7 ) , but not with Sst - Cre neurons ( n = 6 ) . ( F ) Representative smFISH ( Left ) , tSNE plot of 10x Genomics ( Middle ) , quantitative result ( Right ) showed that CLAP was co - localized with Sst - Cre ; Ai9 tdTomato neurons ( n = 6 ) , but not with Gal - Cre neurons ( n = 29 ) . ( G ) Representative smFISH ( Left ) , tSNE plot of 10x Genomics ( Middle ) , quantitative result ( Right ) showed that Gm11549 was co - localized with Slc17a8 - Cre ; Ai9 tdTomato neurons ( n = 5 ) , but not with Mrgprd - Cre neurons ( n = 6 ) . ( H ) Representative smFISH ( Left ) , tSNE plot of 10x Genomics ( Middle ) , quantitative result ( Right ) showed that 2310002F09Rik was co - localized with Slc17a8 - Cre ; Ai9 tdTomato neurons ( n = 6 ) , but not with Slc17a8 - Cre neurons ( n = 6 ) .

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(E) Representative images (Left) of smFISH in DRG section, tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that C530044C16Rik was co-localized with Gal-Cre; Ai9 tdTomato neurons (n = 7), but not with Sst-Cre neurons (n = 6). (F) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that CLAP was co-localized with Sst-Cre; Ai9 tdTomato neurons (n = 6), but not with Gal-Cre neurons (n = 29). (G) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that Gm11549 was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 5), but not with Mrgprd-Cre neurons (n = 6). (H) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that 2310002F09Rik was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 6), but not with Slc17a8-Cre neurons (n = 6).


( I - K ) Representative ( Right ) of double smFISH showed that 2310002F09Rik was rarely co - localized with C530044C16Rik ( I ) , CLAP ( J ) Gm11549 ( K ) . 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 O O O O O O O

(I-K) Representative images (Right) of double smFISH showed that 2310002F09Rik was rarely co-localized with C530044C16Rik (I), CLAP (J) and Gm11549 (K). Scale bar, 10 µm.


( D ) Representative smFISH in DRG sections quantitative results showed that 2310002F09Rik Gm14273 were more enriched in nucleus , whereas CLAP Gm11549 were mainly distributed in cytoplasm . DAPI served as a marker . Scale bar , 10 μm .

(D) Representative smFISH images in DRG sections and quantitative results showed that 2310002F09Rik and Gm14273 were more enriched in the nucleus, whereas CLAP and Gm11549 were mainly distributed in the cytoplasm. DAPI served as a nuclear marker. Scale bar, 10 μm.


( B ) conservation of lincRNA CLAP upstream mRNA Pirt in 9 different species including mouse , human , chimpanzee , rhesus monkey , rat , cow , dog , chicken zebrafish , which were visualized with VISTA browser . CDS , coding . UTR , untranslated region .

(B) Sequence conservation analysis of lincRNA CLAP and upstream mRNA Pirt in 9 different species including mouse, human, chimpanzee, rhesus monkey, rat, cow, dog, chicken and zebrafish, which were visualized with the VISTA browser. CDS, coding sequence. UTR, untranslated region.


( D ) qPCR showed that CLAP was highly expressed in DRG other visceral organs such as small intestine , stomach lung . results are presented as mean ± ( n = 3 ) . ( E ) qPCR showed that expression of CLAP was gradually increased from E13 . 5 to adult DRGs . results are presented as mean ± ( n = 3 ) .

(D) qPCR showed that CLAP was highly expressed in the DRG and other visceral organs such as small intestine, stomach and lung. The results are presented as mean ± SEM (n = 3). (E) qPCR showed that the expression of CLAP was gradually increased from E13.5 to adult DRGs. The results are presented as mean ± SEM (n = 3).


( F ) Representative bright - field showing Sst - cre ; Ai9 neurons for calcium imaging following siCLAP transfection . Scale bar , 10 μm .

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(F) Representative bright-field and fluorescent images showing the Sst-cre; Ai9 neurons for calcium imaging following siCLAP transfection. Scale bar, 10 μm.


( G ) qPCR showed that expression of CLAP was decreased by siRNA treatment in cultured Sst - Cre ; Ai9 DRG neurons . results are presented as mean ± ( n = 3 ) . * * P < 0 . 01 versus siNC .

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(G) qPCR showed that the expression of CLAP was decreased by siRNA treatment in cultured Sst-Cre; Ai9 DRG neurons. The results are presented as mean ± SEM (n = 3). ** P < 0.01 versus siNC.


of Sst - Cre ; Ai9 neurons evoked by histamine ( I ) was decreased by knockdown of CLAP , whereas of these neurons affected by 5 - HT ( J ) , chloroquine ( K ) , capsaicin ( L ) were not changed . results are presented as mean ± ( n = 4 ) . * * P < 0 . 01 versus siNC .

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The percentage of Sst-Cre; Ai9 neurons evoked by histamine (I) was decreased by knockdown of CLAP, whereas the percentage of these neurons affected by 5-HT (J), chloroquine (K), capsaicin (L) were not changed. The results are presented as mean ± SEM (n = 4). ** P < 0.01 versus siNC.


( B ) qPCR showed that CLAP level was significantly decreased by siCLAP in DRG following intrathecal injection . results are presented as mean ± ( siNC = 12 , siCLAP = 15 ) . * * * P < 0 . 001 versus siNC .

(B) qPCR showed that the CLAP level was significantly decreased by siCLAP in the DRG following intrathecal injection. The results are presented as mean ± SEM (siNC = 12, siCLAP = 15). *** P < 0.001 versus siNC.


( C - E ) Knockdown of CLAP reduced itch scratching evoked by histamine ( C ) , but not 5 - HT ( D ) chloroquine ( E ) . results are presented as mean ± ( For histamine 5 - HT , siNC = 12 ， siCLAP = 15 ; for chloroquine , siNC = 5 ， siCLAP = 8 ) . * * P < 0 . 01 versus siNC .

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(C-E) Knockdown of CLAP reduced itch scratching evoked by histamine (C), but not 5-HT (D) and chloroquine (E). The results are presented as mean ± SEM (For histamine and 5-HT, siNC = 12，siCLAP = 15; for chloroquine, siNC = 5，siCLAP = 8 ). ** P < 0.01 versus siNC.


( D ) expression changes of C2 - specific itch - related genes upon CLAP knockdown by - seq . Each dot represents an - seq sample , results are presented as mean ± ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 versus siNC .

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(D) The expression changes of C2-specific and itch-related genes upon CLAP knockdown by RNA-seq. Each dot represents an RNA-seq sample, and results are presented as mean ± SEM (n = 3). * P < 0.05, ** P < 0.01 and *** P < 0.001 versus siNC.


( F ) qPCR showed that downregulated CLAP C2 - specific genes including Sst , Plcb3 Cysltr2 by siCLAP was fully rescued by overexpression of CLAPR in L4 / 5 DRGs of mice . results are presented as mean ± ( siNC , n = 10 ; siCLAP , n = 6 ; siCLAP + CLAPR , n = 12 ) . * P < 0 . 01 versus siNC # P < 0 . 05 , # # P < 0 . 01 versus indicated treatment .

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(F) qPCR showed that downregulated CLAP and C2-specific genes including Sst, Plcb3 and Cysltr2 by siCLAP was fully rescued by overexpression of CLAPR in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 10; siCLAP, n = 6; siCLAP + CLAPR, n = 12). * P < 0.01 versus siNC and # P < 0.05, ## P < 0.01 versus the indicated treatment.