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 .

O O O O B-TISSUE B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-EXP_ASSAY O O B-ORGANISM 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 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 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 .

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

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

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

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 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 .

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

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

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 . 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 .

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

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 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 .

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

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 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

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

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 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 .

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

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 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

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

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 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 .

O O O B-GENEPROD O O B-CELL_LINE O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-CELL_LINE O B-GENEPROD O B-DISEASE O O O O O O O O O B-CELL_LINE O O O B-CELL_LINE O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O

(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 ) 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 .

O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-DISEASE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O

(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 cells cultured under hypoxia for different time intervals . HIF1α accumulation was examined as a positive control .

O O O B-GENEPROD O O O O B-EXP_ASSAY O B-DISEASE I-DISEASE O O O O O O O O O B-GENEPROD O O O O O O O O

(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 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 .

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

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 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 .

O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O

(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 ) 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 ) .

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

(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 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 .

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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 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 .

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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 ) , 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 .

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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 ) 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 .

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(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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 ) .

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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 line , - ) and NBD - Chol ( dotted line , - - - ) from NPs - Chol1 and 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 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 -

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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 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 .

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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 and sacrificed after 24 hrs ( D , left ) or 2 weeks ( D , right ) and 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 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 .

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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 and sacrificed after 12 hrs , showing co - localization of NBD - Chol ( green ) and 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 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 .

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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 ) 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 .

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

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(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 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 .

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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 ) 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 .

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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 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 .

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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 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 )

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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

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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 analysis of extracts of 293T cells transfected with expression vector for Flag - Beclin - 1 and 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 cell line harboring inducible Flag - USP19 was induced with indicated concentration of Doxy for overnight , and the protein was harvested for immunoblot analysis .

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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 levels of the same sample ( B ) were detected by real - time PCR . Bars represented mean ± SEM 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 and treated with rapamycin ( 250 nM ) for 18 h . The 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 ) 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 .

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . The 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 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 .

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

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


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 .

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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 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 .

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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 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 .

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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 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 .

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

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

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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 ) 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 .

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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 , the lysates were analyzed with each antibody .

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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 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 .

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

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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 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 .

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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 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 .

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

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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 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 .

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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 and immunoblot analysis of protein lysates of A549 cells infected with SeV .

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


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 .

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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 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 .

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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 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 .

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

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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 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 .

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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 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 .

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(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 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 .

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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 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 ) .

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

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(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 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 .

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(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 ) 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 .

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(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 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 ) .

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(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 and fluorescent images showing the 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 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 .

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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.


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 .

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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 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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) 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.


( I ) qPCR showed that downregulated Plcb3 by siCLAP was fully rescued by knockdown of MSI2 in L4 / 5 DRGs of mice . The results are presented as mean ± SEM ( siNC , n = 8 ; siCLAP , n = 6 ; siCLAP + siMSI2 , n = 5 ) . * P < 0 . 05 , * * * P < 0 . 001 versus siNC and # # P < 0 . 01 versus the indicated treatment .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR showed that downregulated Plcb3 by siCLAP was fully rescued by knockdown of MSI2 in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 8; siCLAP, n = 6; siCLAP + siMSI2, n = 5). * P < 0.05, *** P < 0.001 versus siNC and ## P < 0.01 versus the indicated treatment.


Human tissue sections were probed with Siglec ‐ 5 Fc and Siglec ‐ 5 R119A Fc to determine the presence of potential sialic acid ‐ independent ligands . Positive signals ( in blue ) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts , as well as in pancreatic tissues .

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

Human tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands. Positive signals (in blue) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts, as well as in pancreatic tissues.