( E ) RT - qPCR - based analysis of expression changes of RFX5 , GBAT2 , WDR74 , HCG15 , POU2F3 , SNORD3B , APOC1P1 , HLA - A , TAP1 , PSMD8 , PDG1 and JUNB genes in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( F ) RT - qPCR - based analysis of expression changes of RFX5 , GBAT2 , PCAT6 , ZNF672 , SSC5D , APOC1P1 , HLA - A , HLA - F and TAP1 genes in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . D

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

(E) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1 and JUNB genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F and TAP1 genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells. D


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , KDM5B , APOC1P1 , and PSMB8 promoters for IgG and H3K4me3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) .

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(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, KDM5B, APOC1P1, and PSMB8 promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) ChIP - qPCRs at RFX5 - GBAT2 , PCAT6 , APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) .

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

(D) ChIP-qPCRs at RFX5-GBAT2, PCAT6, APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , and PSMB8 promoters for IgG and KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) .

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

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, and PSMB8 promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , and H4K8ac peak ( fourth row ) ChIP - Seq data in MDA - MB - 468 for the PRDM10 , TRIM26 , BTN2A1 , PNRC2 , and MR1 genes , visualized by IGV . The green rectangular bars are the locations of the statistically - qualified peak of H3K27ac or H4K8ac in MDA - MB - 468 .

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

(D) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the PRDM10, TRIM26, BTN2A1, PNRC2, and MR1 genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468.


( E ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , and H4K8ac peak ( fourth row ) ChIP - Seq data in MCF7 for the WDR74 , YWHAQ , CDNF / HSPA14 , BTN2A1 , and MR1 genes , visualized by IGV . The turquoise and purple rectangular bars are the locations of the statistically - qualified peak of H3K27ac or H4K8ac in MCF7 , respectively .

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

(E) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the WDR74, YWHAQ, CDNF/HSPA14, BTN2A1, and MR1 genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively.


( D ) RT - qPCR - based analysis of expression changes of DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( E ) RT - qPCR - based analysis of expression changes of KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( F ) RT - qPCR - based analysis of expression changes of DVL3 in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( G ) RT - qPCR - based analysis of expression changes of KMT2D in MCF7 ( NTC , shA DVL3 , and shB DVL3 ) cells . ( H

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

(D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (H


( H ) Immunofluorescence of KMT2D localization in MDA - MB - 468 cells . The cells were probed with KMT2D antibody ( red ) . The nucleus was stained with DAPI ( blue ) and the actin filaments ( green ) were stained with Phalloidin . Merge of KMT2D ( red ) and nuclear staining ( blue ) is shown as KMT2D / DAPI , merge of KMT2D ( red ) and actin ( green ) is shown as KMT2D / ACTIN , and , merge of KMT2D ( red ) , nuclear staining ( blue ) and actin ( green ) is shown as MERGE for NTC , shA DVL3 and shB DVL3 cells . Scale bar :

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

(H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm


( C ) IVIS images showing representative tumors generated by injecting luciferase - expressing MDA - MB - 468 cells ( NTC , shA DVL3 or shB DVL3 ) in the mammary fat pad of SCID mice . Color scale ( luminescent signal intensity ) : blue , least intense signal ; red , most intense signal . ( D ) Quantification of luminescence changes in tumors generated by injecting luciferase - expressing MDA - MB - 468 cells ( NTC , shA DVL3 or shB DVL3 ) in the mammary fat pad of SCID mice , Average luminescence was used to measure volume of tumor ; n ≥ 9 . (

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

(C) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9. (


( G ) IVIS images showing representative tumors generated by injecting luciferase - expressing MCF7 cells ( NTC , shA DVL3 ) in the mammary fat pad of SCID mice . Color scale ( luminescent signal intensity ) : blue , least intense signal ; red , most intense signal . ( H ) Quantification of luminescence changes in tumors generated by injecting luciferase - expressing MCF7 cells ( NTC , shA DVL3 ) in the mammary fat pad of SCID mice . Average luminescence was used to measure volume of tumor . n = 7 .

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

(G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.


( A ) Immunofluorescence of HA - tagged DVL3 localization in MDA - MB - 468 cells . The cells were probed with an HA antibody ( red ) . The nucleus was stained with DAPI ( blue ) and the actin filaments ( green ) were stained with Phalloidin . Merge of actin ( green ) and nuclear staining ( blue ) is shown as ACTIN / DAPI , merge of DVL3 ( red ) and nuclear staining ( blue ) is shown as HA / DAPI , merge of DVL3 ( red ) and actin ( green ) is shown as HA / ACTIN , and , merge of DVL3 ( red ) , nuclear staining ( blue ) and actin ( green ) is shown as MERGE for empty vector ( EV ) , HA - DVL3 - WT and HA - DVL3 - NESm transient transfected cells . Scale bar : 10µm .

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

(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.


( B ) RT - qPCR - based analysis of expression changes of DVL3 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( C ) RT - qPCR - based analysis of expression changes of KMT2D in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( D ) RT - qPCR - based analysis of expression changes of ARAP1 , RFX5 , GBAT2 , WDR74 , SNORD3D , HAUS5 , TAP1 , HLA - A , PDG1 , and , MR1 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) cells . ( E

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

(B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (D) RT-qPCR-based analysis of expression changes of ARAP1 , RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1, and, MR1 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (E


( A ) Representative clones screened for CD24 expression under regulation of tetracycline . The cells were exposed to 1 µg / ml tetracycline for 48 h . 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti - CD24 SWA11 mAb . The membrane was reprobed with anti - tubulin antibody to assess the uniformity of sample loading .

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

(A) Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 µg/ml tetracycline for 48 h. 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti-CD24 SWA11 mAb. The membrane was reprobed with anti-tubulin antibody to assess the uniformity of sample loading.


( D ) Western blot analysis of EXO - CD24 exosomes . Tetracycline ( Tet ) was used to induce CD24 expression in the cell line from which exosomes are derived . HSP70 was used as an exosomal marker .

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

(D) Western blot analysis of EXO-CD24 exosomes. Tetracycline (Tet) was used to induce CD24 expression in the cell line from which exosomes are derived. HSP70 was used as an exosomal marker.


( C ) Western blot analysis of EXO - mCD24 exosomes . HSP70 was used as an exosomal marker .

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(C) Western blot analysis of EXO-mCD24 exosomes. HSP70 was used as an exosomal marker.


( B ) respiratory rate and ( C ) Blood oxygen ( SpO2 ) and before treatment ( Day 1 , measured before administration of first dose ) and after treatment ( Day 7 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for Blood oxygen ( SpO2 ) and respiratory rate . these are presented graphically via box plots before and after treatment .

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

(B) respiratory rate and (C) Blood oxygen (SpO2) and before treatment (Day 1, measured before administration of first dose) and after treatment (Day 7). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for Blood oxygen (SpO2) and respiratory rate. these are presented graphically via box plots before and after treatment.


Systemic CRP levels . Values are normalized against baseline value ( Day 0 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for CRP levels . these are presented graphically via box plots before and after treatment ( n = 35 patients )

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Systemic CRP levels. Values are normalized against baseline value (Day 0). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for CRP levels. these are presented graphically via box plots before and after treatment (n=35 patients)


( B ) Systemic cytokine levels . Cytokine levels were measured during ( Day 3 / 4 ) or after ( Day 7 / 8 ) treatment with EXO - CD24 using Quantibody Multiplex ELISA Array . Values are normalized against baseline value ( Day 0 ) . Samples from 11 patients were analysed . Results from 8 patients are shown ( results for 3 patients are not shown because one or more baseline results were below detection level ) . Box plot of Systemic cytokine levels by days after treatment ( minimum , maximum , median , first quartile , third quartile ) .

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

(B) Systemic cytokine levels. Cytokine levels were measured during (Day 3/4) or after (Day 7/8) treatment with EXO-CD24 using Quantibody Multiplex ELISA Array. Values are normalized against baseline value (Day 0). Samples from 11 patients were analysed. Results from 8 patients are shown (results for 3 patients are not shown because one or more baseline results were below detection level). Box plot of Systemic cytokine levels by days after treatment (minimum, maximum, median, first quartile, third quartile).


( C ) During the course of EXO - CD24 treatment and follow - up , cytokine levels were measured for the rest of the patients ( n = 24 patients ) .

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(C) During the course of EXO-CD24 treatment and follow-up, cytokine levels were measured for the rest of the patients (n=24 patients).


( D ) Monitoring systemic IL - 6 cytokine level following EXO - CD24 treatment . Samples from 17 patients , representing the 4 experimental groups , were analysed using the BD ™ Cytometric Bead Array ( CBA ) .

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(D) Monitoring systemic IL-6 cytokine level following EXO-CD24 treatment. Samples from 17 patients, representing the 4 experimental groups, were analysed using the BD™ Cytometric Bead Array (CBA).


A . GFP - Snc1 - PEM protein accumulates in atg1Δ , atg8Δ , and atg11Δ mutant cells . Lysates were prepared from wild type ( WT ) , ypt1 - 1 ( for comparison ) , atg1Δ , atg8Δ , and atg11Δ mutant cells transformed with a 2μ plasmid expressing GFP - Snc1 - PEM from the TPI promoter . The level of GFP - Snc1 - PEM was determined using immunoblot analysis with anti - GFP antibodies . The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control .

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A. GFP-Snc1-PEM protein accumulates in atg1Δ, atg8Δ, and atg11Δ mutant cells. Lysates were prepared from wild type (WT), ypt1-1 (for comparison), atg1Δ, atg8Δ, and atg11Δ mutant cells transformed with a 2μ plasmid expressing GFP-Snc1-PEM from the TPI promoter. The level of GFP-Snc1-PEM was determined using immunoblot analysis with anti-GFP antibodies. The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control.


B . GFP - Snc1 - PEM protein accumulates in aberrant ER structures in atg1Δ , atg8Δ , and atg11Δ mutant cells . The ER - marker Sec61 was tagged with mCherry in strains from panel A , and the cells were examined by live - cell microscopy . Shown from left to right : DIC , GFP , mCherry , merge , % cells with intracellular GFP - Snc1 - PEM ( number of cells with internal GFP - Snc1 - PEM / number of cells visualized ) , and % cells in which intra - cellular Snc1 - PEM co - localizes with Sec61 .

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B. GFP-Snc1-PEM protein accumulates in aberrant ER structures in atg1Δ, atg8Δ, and atg11Δ mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61.


C . UPR is induced in atg1Δ , atg8Δ , and atg11Δ mutant cells . Cells from panel A were transformed with a second plasmid that expresses β - gal from a UPR promoter . UPR was determined and expressed as % of the WT response .

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C. UPR is induced in atg1Δ, atg8Δ, and atg11Δ mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response.


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level (

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D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level ( D ) , intra - cellular accumulation of GFP - Snc1 - PEM , ( E ) ,

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D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E),


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level ( D ) , intra - cellular accumulation of GFP - Snc1 - PEM , ( E ) , and induction of the UPR response ( F ) . Wild type ( WT ) , atg19Δ , atg11Δ , atg32Δ , and atg36Δ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for panels A - C , respectively . E . Shown from left to right : DIC , GFP , and % cells with intracellular Snc1 - PEM structures . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E), and induction of the UPR response (F). Wild type (WT), atg19Δ, atg11Δ, atg32Δ, and atg36Δ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. E. Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) ,

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A),


A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B )

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B)


A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B ) , and induction of the UPR response ( C , atg1 ∆ is shown as a positive control ) .

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B), and induction of the UPR response (C,atg1∆ is shown as a positive control).


D - G . Atg9 is epistatic to Atg11 in macro - ER - phagy . ATG9 was deleted in wild type and atg11 ∆ mutant cells and the effects of overexpression of GFP - Snc1 - PEM were determined in the single and double mutants as described in Fig 1 legend . D . Deletion of ATG9 in wild type ( WT ) or atg11 ∆ mutant cells results in an increase of GFP - Snc1 - PEM protein level similar to the increase in atg11 ∆ mutant cells .

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D-G. Atg9 is epistatic to Atg11 in macro-ER-phagy. ATG9 was deleted in wild type and atg11∆ mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in Fig 1 legend. D. Deletion of ATG9 in wild type (WT) or atg11∆ mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in atg11∆ mutant cells.


E . Deletion of ATG9 in wild type or atg11 ∆ mutant cells results in an increase of intracellular GFP - Snc1 - PEM fluorescence . However , only ~ 20 % of the atg9 ∆ single - , and atg9 ∆ atg11 ∆ double - mutant cells accumulate GFP - Snc1 - PEM in aberrant structures , as compared with ~ 75 % of atg11 ∆ mutant cells . Shown from top to bottom : DIC , GFP , % cells with aberrant intracellular GFP - Snc1 - PEM structures , ratio of GFP - Snc1 - PEM fluorescence inside / PM ( 30 cells were analyzed for each strain ) .

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E. Deletion of ATG9 in wild type or atg11∆ mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the atg9∆ single-, and atg9∆ atg11∆ double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of atg11∆ mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain).


F . UPR is induced in atg11 ∆ , but not in atg9 ∆ single - and atg9 ∆ atg11 ∆ double - mutant cells overexpressing GFP - Snc1 - PEM .

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F. UPR is induced in atg11∆, but not in atg9∆ single- and atg9∆ atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM.


G . UPR can be induced in atg9 ∆ single - , and atg9 atg11 ∆ double - mutant cells overexpressing GFP - Snc1 - PEM by tunicamycin . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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G. UPR can be induced in atg9∆ single-, and atg9 atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . ATG9 was deleted in ypt1 - 1 , WT and sec12ts mutant cells and the effect of overexpression of GFP - Snc1 - PEM was determined in single and double mutant cells . Experiments were performed as described for Fig 1A - 1C , respectively . A . Snc1 - PEM is increased ~ 3 fold in atg9 ∆ and sec12ts as compared to ~ 20 fold in ypt1 - 1 mutant cells . Importantly , atg9 ∆ is epstatic to the ypt1 - 1 , but not to the sec12ts , mutation . Left , immuno - blot analysis , increase of the protein level in mutant versus the WT cells is shown under the blot ; right , a bar graph summarizing the quantified

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A-C.ATG9 was deleted in ypt1-1, WT and sec12ts mutant cells and the effect of overexpression of GFP-Snc1-PEM was determined in single and double mutant cells. Experiments were performed as described for Fig 1A-1C, respectively. A. Snc1-PEM is increased ~3 fold in atg9∆ and sec12ts as compared to ~20 fold in ypt1-1 mutant cells. Importantly, atg9∆ is epstatic to the ypt1-1, but not to the sec12ts, mutation. Left, immuno-blot analysis, increase of the protein level in mutant versus the WT cells is shown under the blot; right, a bar graph summarizing the quantified data


. Whereas deletion of ATG9 in ypt1 - 1 mutant cells results in three fold lower accumulation of GFP - Snc1 - PEM in aberrant structures ( 85 % to 27 % ) , its deletion in sec12ts mutant cells results in two fold increased accumulation ( ~ 40 % to ~ 80 % ) . Shown from top to bottom : DIC , GFP , and % cells with intracellular Snc1 - PEM structures .

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. Whereas deletion of ATG9 in ypt1-1 mutant cells results in three fold lower accumulation of GFP-Snc1-PEM in aberrant structures (85% to 27%), its deletion in sec12ts mutant cells results in two fold increased accumulation (~40% to ~80%). Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.


C . Deletion of ATG9 in ypt1 - 1 mutant cells overexpressing Snc1 - PEM results in ~ 3 . 5 lower UPR ( p - value < 0 . 0005 ) , but a slightly increased UPR in sec12ts mutant cells ( p - value = 0 . 05 ) .

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C. Deletion of ATG9 in ypt1-1 mutant cells overexpressing Snc1-PEM results in ~3.5 lower UPR (p-value<0.0005), but a slightly increased UPR in sec12ts mutant cells (p-value = 0.05).


D . Atg9 is present on aberrant ER structures that accumulate in ypt1 - 1 mutant cells . WT and ypt1 - 1 mutant cells expressing Atg9 - mCherry from the chromosome and GFP - Snc1 - PEM from a 2μ plasmid were analyzed by live - cell microscopy . Whereas in WT cells ( top ) , the GFP - Snc1 - PEM localizes to the cell membrane and Atg9 - mCherry to intracellular puncta , the two proteins co - localize in 100 % of the ypt1 - 1 mutant cells ( bottom ) that accumulate intracellular GFP - Snc1 - PEM structures ( ~ 80 % ) . Shown from left to right : DIC , GFP , mCherry , merge , % cells with intracellular Snc1 - PEM , and % cells with co - localization ( number of cells with observed phenotype / total number of cells analyzed ) .

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D. Atg9 is present on aberrant ER structures that accumulate in ypt1-1 mutant cells. WT and ypt1-1 mutant cells expressing Atg9-mCherry from the chromosome and GFP-Snc1-PEM from a 2μ plasmid were analyzed by live-cell microscopy. Whereas in WT cells (top), the GFP-Snc1-PEM localizes to the cell membrane and Atg9-mCherry to intracellular puncta, the two proteins co-localize in 100% of the ypt1-1 mutant cells (bottom) that accumulate intracellular GFP-Snc1-PEM structures (~80%). Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular Snc1-PEM, and % cells with co-localization (number of cells with observed phenotype / total number of cells analyzed).


E . GFP - Snc1 - PEM is delivered to the vacuole for degradation in sec12ts mutant cells . Accumulation of GFP - Snc1 - PEM in vacuoles of sec12ts mutant cells , with and without deletion of the vacuolar protease Pep4 , was determined using the FM4 - 64 dye , which labels the vacuolar membrane . Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP - Snc1 - PEM accumulates inside the vacuole ( from 8 % to 100 % ) . Shown from left to right : DIC , GFP , FM4 - 64 ( vacuolar membrane ) , merge , % cells with aberrant GFP - Snc1 - PEM structures , % cells with GFP - Snc1 - PEM outside vacuole , and % cells with GFP - Snc1 - PEM inside the vacuole . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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E. GFP-Snc1-PEM is delivered to the vacuole for degradation in sec12ts mutant cells. Accumulation of GFP-Snc1-PEM in vacuoles of sec12ts mutant cells, with and without deletion of the vacuolar protease Pep4, was determined using the FM4-64 dye, which labels the vacuolar membrane. Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP-Snc1-PEM accumulates inside the vacuole (from 8% to 100%). Shown from left to right: DIC, GFP, FM4-64 (vacuolar membrane), merge, % cells with aberrant GFP-Snc1-PEM structures, % cells with GFP-Snc1-PEM outside vacuole, and % cells with GFP-Snc1-PEM inside the vacuole. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . The ypt1 - 1 mutation is epistatic to vps21 ∆ in ER - phagy . yDsRed - Snc1 - PEM was overexpressed in WT , vps21 ∆ , ypt1 - 1 and ypt1 - 1vps21 ∆ double mutant cells that also expressed the autophagosomal marker yEGFP - Atg8 . Cells were analyzed by live - cell microscopy . Shown from left to right : DIC , DsRed , GFP , merge , % cells Atg8 dots , number of Atg8 dots per cell , and % cells in which the Atg8 dots co - localize with Snc1 - PEM . About 50 % of WT and 85 % of vps21 ∆ mutant cells contain ~ 1 dot of Atg8 representing the AP . Importantly , in ~ 70 % of the vps21 ∆ mutant cells Snc1 - PEM co - localizes with the APs , as compared to ~ 4 % in WT cells . In contrast , ~ 90 % ypt1 - 1 and ypt1 - 1vps21 ∆ mutant cells contain three APs per cell , and Snc1 - PEM does not co - localize with them . Arrows point to co - localization ; arrowheads point to either Atg8 dots or GFP - Snc1 - PEM that do not co - localize .

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A. The ypt1-1 mutation is epistatic to vps21∆ in ER-phagy. yDsRed-Snc1-PEM was overexpressed in WT, vps21∆, ypt1-1 and ypt1-1vps21∆ double mutant cells that also expressed the autophagosomal marker yEGFP-Atg8. Cells were analyzed by live-cell microscopy. Shown from left to right: DIC, DsRed, GFP, merge, % cells Atg8 dots, number of Atg8 dots per cell, and % cells in which the Atg8 dots co-localize with Snc1-PEM. About 50% of WT and 85% of vps21∆ mutant cells contain ~1 dot of Atg8 representing the AP. Importantly, in ~70% of the vps21∆ mutant cells Snc1-PEM co-localizes with the APs, as compared to ~4% in WT cells. In contrast, ~90% ypt1-1 and ypt1-1vps21∆ mutant cells contain three APs per cell, and Snc1-PEM does not co-localize with them. Arrows point to co-localization; arrowheads point to either Atg8 dots or GFP-Snc1-PEM that do not co-localize.


The following effects of overexpression of GFP - Snc1 - PEM in WT and ypt1 - 1 mutant cells , without and with ire1Δ or hac1Δ , were analyzed as described for Fig 1A - 1C , respectively : protein level ( B ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( C ) , and UPR induction ( D ) . B . Deletion of either Ire1 or Hac1 does not affect the level of Snc1 - PEM accumulation in WT or ypt1 - 1 mutant cells .

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The following effects of overexpression of GFP-Snc1-PEM in WT and ypt1-1 mutant cells, without and with ire1Δ or hac1Δ, were analyzed as described for Fig 1A-1C, respectively: protein level (B), accumulation of GFP-Snc1-PEM in aberrant structures (C), and UPR induction (D). B. Deletion of either Ire1 or Hac1 does not affect the level of Snc1-PEM accumulation in WT or ypt1-1 mutant cells.


C . Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1 - 1 mutant cells that accumulate aberrant intra - cellular Snc1 - PEM . Shown from top to bottom : DIC , GFP , and % cells with intracellular Snc1 - PEM structures .

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C. Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1-1 mutant cells that accumulate aberrant intra-cellular Snc1-PEM. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.


D . Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1 - 1 mutant cells . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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D. Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1-1 mutant cells. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . Overexpressed Snq2 - GFP accumulates in the ER and induces UPR in ypt1 - 1 mutant cells . Snq2 - yEGFP was overexpressed in WT and ypt1 - 1 mutant cells and the following effects were analyzed as described for Fig 1A - 1C , respectively : increase in the level of Snq2 - yEGFP protein ( A )

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A)


A - C . Overexpressed Snq2 - GFP accumulates in the ER and induces UPR in ypt1 - 1 mutant cells . Snq2 - yEGFP was overexpressed in WT and ypt1 - 1 mutant cells and the following effects were analyzed as described for Fig 1A - 1C , respectively : increase in the level of Snq2 - yEGFP protein ( A ) , accumulation of Snq2 - yEGFP in aberrant ER structures ( B ) ,

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B),


A - C . Overexpressed Snq2 - GFP accumulates in the ER and induces UPR in ypt1 - 1 mutant cells . Snq2 - yEGFP was overexpressed in WT and ypt1 - 1 mutant cells and the following effects were analyzed as described for Fig 1A - 1C , respectively : increase in the level of Snq2 - yEGFP protein ( A ) , accumulation of Snq2 - yEGFP in aberrant ER structures ( B ) , and induction of the UPR response ( C , GFP - Snc1 - PEM is shown for comparison ) .

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B), and induction of the UPR response (C, GFP-Snc1-PEM is shown for comparison).


D - F . Synergistic effect of co - overexpression of Snq2 - GFP with DsRed - Snc1 - PEM in atg11 ∆ mutant cells . WT ( left ) and atg11 ∆ mutant cells ( right ) were transformed with plasmids for overexpression of Snq2 , Snc1 - PEM or both Snq2 and Snc1 - PEM . D . Immunoblot analysis and quantification . Shown from top to bottom : Snq2 - yEGFP ( using anti - GFP antibodies ) , Ds - Red - Snc1 - PEM ( using anti - Snc1 antibodies ) , G6PDH ( loading control ) , and a bar graph showing fold increase of Snq2 - yEGFP ( green ) and Ds - Red - Snc1 - PEM ( red ) in atg11 ∆ mutant cells over WT .

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D-F. Synergistic effect of co-overexpression of Snq2-GFP with DsRed-Snc1-PEM in atg11∆ mutant cells. WT (left) and atg11∆ mutant cells (right) were transformed with plasmids for overexpression of Snq2, Snc1-PEM or both Snq2 and Snc1-PEM. D. Immunoblot analysis and quantification. Shown from top to bottom: Snq2-yEGFP (using anti-GFP antibodies), Ds-Red-Snc1-PEM (using anti-Snc1 antibodies), G6PDH (loading control), and a bar graph showing fold increase of Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) in atg11∆ mutant cells over WT.


E . Accumulation of macro - ER - phagy cargos in aberrant intracellular structures using live - cells microscopy . Shown from left to right : DIC , GFP , DsRed and Merge . Shown from top to bottom : Snq2 , Snc1 - PEM , Snq2 + Snc1 - PEM , and % cells with co - localization ( relevant only for co - overexpression ) ( % cells with ER - phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig ) . F . Fluorescence level of intracellular structures ( ratio over PM ) in atg11 ∆ mutant cells . The bar graph shows Snq2 - yEGFP ( green ) and Ds - Red - Snc1 - PEM ( red ) fluorescence ( 20 cells were analyzed for each strain ) . When co - overexpressed in atg11 ∆ mutant cells , the fluorescence level of either protein accumulating in aberrant structures is ~ 5 fold higher than when overexpressed individually . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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E. Accumulation of macro-ER-phagy cargos in aberrant intracellular structures using live-cells microscopy. Shown from left to right: DIC, GFP, DsRed and Merge. Shown from top to bottom: Snq2, Snc1-PEM, Snq2+Snc1-PEM, and % cells with co-localization (relevant only for co-overexpression) (% cells with ER-phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig). F. Fluorescence level of intracellular structures (ratio over PM) in atg11∆ mutant cells. The bar graph shows Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) fluorescence (20 cells were analyzed for each strain). When co-overexpressed in atg11∆ mutant cells, the fluorescence level of either protein accumulating in aberrant structures is ~5 fold higher than when overexpressed individually. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . A diagram showing two groups of ER - resident proteins : those that become macro - ER - phagy cargos ( in green ) , and those that do not ( in blue or grey ) . Like overexpressed Snc1 - PEM or Snq2 , the ER - resident membrane proteins Sec61 ( translocon subunit ) and Hmg1 ( sterol biogenesis ) are transported to the vacuole via macro - ER - phagy . In contrast , the ER - to - Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co - transported to the lysosome through macro - ER - phagy . Four or six different strains were used for this analysis : WT ( YPT1 ) , pep4 ∆ , and pep4 prb1 ∆ , ypt1 - 1 , ypt1 - 1pep4 ∆ , and ypt1 - 1pep4 ∆ prb1 ∆ . In each strain , one ER - resident protein was tagged at its C - terminus ( except for Kar2 ) . ( The pep4 ∆ strains were not used in all experiments because they do not show the full defect ) . The level of ER - resident proteins was determined by immunoblot analysis in cells that either do not ( B - D ) or do overexpress GFP - Snc1 - PEM ( F - H ) . B - C . Sec61 - 3xHA ( B ) and Sec13 - 3x HA ( C ) expressing cells ( 2 independent un - transformed colonies ) were tested by immunoblot analysis ( using anti - HA antibodies ) . Shown from top to bottom : strain genotype , HA - tagged protein , G6PDH ( loading control ) , quantification of HA - tagged protein expressed as average fold of WT ( p - value = 0 . 025 for Sec61 ) .

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A. A diagram showing two groups of ER-resident proteins: those that become macro-ER-phagy cargos (in green), and those that do not (in blue or grey). Like overexpressed Snc1-PEM or Snq2, the ER-resident membrane proteins Sec61 (translocon subunit) and Hmg1 (sterol biogenesis) are transported to the vacuole via macro-ER-phagy. In contrast, the ER-to-Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co-transported to the lysosome through macro-ER-phagy. Four or six different strains were used for this analysis: WT (YPT1), pep4∆, and pep4 prb1∆, ypt1-1, ypt1-1pep4∆, and ypt1-1pep4∆ prb1∆. In each strain, one ER-resident protein was tagged at its C-terminus (except for Kar2). (The pep4∆ strains were not used in all experiments because they do not show the full defect). The level of ER-resident proteins was determined by immunoblot analysis in cells that either do not (B-D) or do overexpress GFP-Snc1-PEM (F-H). B-C. Sec61-3xHA (B) and Sec13-3x HA (C) expressing cells (2 independent un-transformed colonies) were tested by immunoblot analysis (using anti-HA antibodies). Shown from top to bottom: strain genotype, HA-tagged protein, G6PDH (loading control), quantification of HA-tagged protein expressed as average fold of WT (p-value = 0.025 for Sec61).


D . Summary of immunoblot analyses of ER - resident proteins level in cells that do not overexpress GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots and quantification are shown in Fig 6B - 6C and S6A - S6C Fig . In YPT1 cells , the levels of Sec61 and Hmg1 ( green ) are slightly increased ( 14 and 38 % , respectively ) in strains defective in vacuolar proteolysis ; the levels of Sec12 , Sec13 and Kar2 are not increased ( blue and grey ) . The levels of Sec61 and Hmg1 ( green ) are increased by 2 - 2 . 5 fold in ypt1 - 1 mutant cells when compared to WT cells , regardless if they are defective in vacuolar proteolysis or not . In contrast , the levels of Sec12 , Sec13 and Kar2 are only slightly increased ( blue and grey ) .

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D. Summary of immunoblot analyses of ER-resident proteins level in cells that do not overexpress GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6B-6C and S6A-S6C Fig. In YPT1 cells, the levels of Sec61 and Hmg1 (green) are slightly increased (14 and 38%, respectively) in strains defective in vacuolar proteolysis; the levels of Sec12, Sec13 and Kar2 are not increased (blue and grey). The levels of Sec61 and Hmg1 (green) are increased by 2-2.5 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey).


E . The level of the ER - resident protein Sec61 is similar whether GFP - Snc1 - PEM is overexpressed or not . Wild - type cells were transformed with a 2μ plasmid , either empty or for overexpression of GFP - Snc1 - GEM ( 2 independent transformants ) . Shown from top to bottom : plasmid , Sec61 , G6PDH ( loading control ) , GFP - Snc1 - PEM , and quantification of Sec61 expressed as average fold of WT with empty plasmid .

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E. The level of the ER-resident protein Sec61 is similar whether GFP-Snc1-PEM is overexpressed or not. Wild-type cells were transformed with a 2μ plasmid, either empty or for overexpression of GFP-Snc1-GEM (2 independent transformants). Shown from top to bottom: plasmid, Sec61, G6PDH (loading control), GFP-Snc1-PEM, and quantification of Sec61 expressed as average fold of WT with empty plasmid.


F - G . Sec61 - 3xHA ( F ) and Sec13 - 3xHA ( G ) expressing cells were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM . Cell lysates were tested by immunoblot analysis ( using ant - HA and anti - GFP antibodies ) . Shown from top to bottom : strain genotype , the specific ER - resident protein tested , quantification of the ER - resident protein bands expressed as average fold of WT , GFP - Snc1 - PEM , quantification of the GFP - Snc1 - PEM bands expressed as average fold of WT , and G6PDH ( loading control ) .

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F-G. Sec61-3xHA (F) and Sec13-3xHA (G) expressing cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM. Cell lysates were tested by immunoblot analysis (using ant-HA and anti-GFP antibodies). Shown from top to bottom: strain genotype, the specific ER-resident protein tested, quantification of the ER-resident protein bands expressed as average fold of WT, GFP-Snc1-PEM, quantification of the GFP-Snc1-PEM bands expressed as average fold of WT, and G6PDH (loading control).


H . Summary of immunoblot analysis of ER - resident proteins level in cells overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots and quantification are shown in Fig 6F and 6G and S6F - S6H Fig . In YPT1 cells , like Snc1 - PEM ( black ) , the levels of Hmg1 and Sec61 ( green ) are increased > 10 fold in strains defective in vacuolar proteolysis . In contrast , the levels of Sec12 , Sec13 and Kar2 are not changed ( blue and grey ) . Like Snc1 - PEM ( black ) , the protein levels of Hmg1 and Sec61 ( green ) are increased ~ 15 fold in ypt1 - 1 mutant cells when compared to WT cells , regardless if they are defective in vacuolar proteolysis or not . In contrast , the levels of Sec12 , Sec13 and Kar2 are only slightly increased ( blue and grey ) . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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H. Summary of immunoblot analysis of ER-resident proteins level in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6F and 6G and S6F-S6H Fig. In YPT1 cells, like Snc1-PEM (black), the levels of Hmg1 and Sec61 (green) are increased >10 fold in strains defective in vacuolar proteolysis. In contrast, the levels of Sec12, Sec13 and Kar2 are not changed (blue and grey). Like Snc1-PEM (black), the protein levels of Hmg1 and Sec61 (green) are increased ~15 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey). +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . In cells defective in vacuolar proteolysis ( pep4 ∆ prb1 ∆ ) , Hmg1 co - localizes with 70 % of intra - cellular Snc1 - PEM , but Sec13 does not ( at least 36 cells were analyzed for each strain ) . No co - localization was observed for resident ER proteins and overexpressed Snc1 - PEM in WT cells ( PEP4PRB1 ) . Hmg1 ( left ) and Sec13 ( right ) were tagged with mCherry at their C - terminus in PEP4PRB1 ( WT ) and pepb4 ∆ prb1 ∆ mutant cells . Cells were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM and analyzed by live - cell microscopy . Shown from top to bottom : DIC , GFP , mCherry , merge ( yellow ) , % cells with intra - cellular Snc1 - PEM , and % co - localization of the ER protein with intra - cellular Snc1 - PEM .

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A. In cells defective in vacuolar proteolysis (pep4∆ prb1∆), Hmg1 co-localizes with 70% of intra-cellular Snc1-PEM, but Sec13 does not (at least 36 cells were analyzed for each strain). No co-localization was observed for resident ER proteins and overexpressed Snc1-PEM in WT cells (PEP4PRB1). Hmg1 (left) and Sec13 (right) were tagged with mCherry at their C-terminus in PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells. Cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM and analyzed by live-cell microscopy. Shown from top to bottom: DIC, GFP, mCherry, merge (yellow), % cells with intra-cellular Snc1-PEM, and % co-localization of the ER protein with intra-cellular Snc1-PEM.


B . The ER - resident protein Hmg1 is delivered to the vacuole with GFP - Snc1 - PEM . The vacuole of PEP4PRB1 ( WT ) and pepb4 ∆ prb1 ∆ mutant cells expressing Hmg1 - mCherry and overexpressing GFP - Snc1 - PEM ( see Panel A , left ) was stained with CMAC - Arg ( blue ) , and the cells were analyzed by live - cell microscopy . In WT cells ( top ) , there is no intracellular Snc1 - PEM and the red - labeled ER is distinguished from the blue vacuole . In contrast , in ~ 75 % pep4 ∆ prb1 ∆ mutant cells ( bottom ) , which are defective in vacuolar proteolysis , Hmg1 ( red ) co - localizes with Snc1 - PEM ( green ) in the vacuole ( blue ) . Shown from left to right : DIC , GFP , mCherry , blue , merge ( white shows merge of the three colors ) , % cells with intracellular Snc1 - PEM , % cells in which Hmg1 with Snc1 - PEM co - localize , and % co - localization of the two proteins with the vacuolar dye ( 38 cells were analyzed for each strain ) .

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B. The ER-resident protein Hmg1 is delivered to the vacuole with GFP-Snc1-PEM. The vacuole of PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells expressing Hmg1-mCherry and overexpressing GFP-Snc1-PEM (see Panel A, left) was stained with CMAC-Arg (blue), and the cells were analyzed by live-cell microscopy. In WT cells (top), there is no intracellular Snc1-PEM and the red-labeled ER is distinguished from the blue vacuole. In contrast, in ~75% pep4∆ prb1∆ mutant cells (bottom), which are defective in vacuolar proteolysis, Hmg1 (red) co-localizes with Snc1-PEM (green) in the vacuole (blue). Shown from left to right: DIC, GFP, mCherry, blue, merge (white shows merge of the three colors), % cells with intracellular Snc1-PEM, % cells in which Hmg1 with Snc1-PEM co-localize, and % co-localization of the two proteins with the vacuolar dye (38 cells were analyzed for each strain).


C . > 80 % of ypt1 - 1 mutant cells , regardless if they are PEP4PRB1 or pep4 ∆ prb1 ∆ , accumulate aberrant GFP - Snc1 - PEM structures . Hmg1 co - localizes with > 70 % of these structures , whereas Sec13 co - localizes with only ~ 15 % of these structures ( 36 - 43 cells were analyzed for each strain ) . The experiment was done as described for Panel A .

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C. >80% of ypt1-1 mutant cells, regardless if they are PEP4PRB1 or pep4∆ prb1∆, accumulate aberrant GFP-Snc1-PEM structures. Hmg1 co-localizes with >70% of these structures, whereas Sec13 co-localizes with only ~15% of these structures (36-43 cells were analyzed for each strain). The experiment was done as described for Panel A.


D . Summary of microscopy analysis of ER - resident proteins co - localization with GFP - Snc1 - PEM in cells overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4Δ prb1Δ ( left ) , and ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4Δ prb1Δ ( right ) . Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig . In YPT1 cells , whereas > 60 % of Hmg1 and Sec61 ( green ) co - localize with Snc1 - PEM in strains defective in vacuolar proteolysis , Sec12 , Sec13 and Kar2 are not changed significantly ( blue and grey ) . Hmg1 and Sec61 ( green ) co - localize with Snc1 - PEM in > 70 % of ypt1 - 1 and > 80 % of ypt1 - 1pep4Δ prb1Δ mutant cells , whereas Sec13 and Kar2 ( blue and grey ) do so in < 15 % of the cells . The level of Sec12 co - localization with Snc1 - PEM is intermediate , ~ 38 % in ypt1 - 1 and ypt1 - 1pep4Δ prb1Δ . Error bars represent STDEV . Results in panels A and B represent at least two independent experiments .

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D. Summary of microscopy analysis of ER-resident proteins co-localization with GFP-Snc1-PEM in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4Δ prb1Δ (left), and ypt1-1PEP4PRB1 vs ypt1-1pep4Δ prb1Δ (right). Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig. In YPT1 cells, whereas >60% of Hmg1 and Sec61 (green) co-localize with Snc1-PEM in strains defective in vacuolar proteolysis, Sec12, Sec13 and Kar2 are not changed significantly (blue and grey). Hmg1 and Sec61 (green) co-localize with Snc1-PEM in >70% of ypt1-1 and >80% of ypt1-1pep4Δ prb1Δ mutant cells, whereas Sec13 and Kar2 (blue and grey) do so in <15% of the cells. The level of Sec12 co-localization with Snc1-PEM is intermediate, ~38% in ypt1-1 and ypt1-1pep4Δ prb1Δ. Error bars represent STDEV. Results in panels A and B represent at least two independent experiments.


A . Top : DDX42 KO and CTRL KO U87 - MG / CD4 / CXCR4 / Cas9 / Firefly cells were generated using 3 sgRNAs and 4 non - targeting sgRNAs , respectively ( for CTRL , the average of data obtained with 4 cell populations is shown ) . Cells were treated or not with IFN 24 h prior to infection with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) . Relative luminescence results for IFN - treated and - untreated conditions are shown . Two - way ANOVA on log - transformed data with Sidak ' s test . Bottom : Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42 - depleted populations ; Actin served as a loading control .

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A. Top: DDX42 KO and CTRL KO U87-MG/CD4/CXCR4/Cas9/Firefly cells were generated using 3 sgRNAs and 4 non-targeting sgRNAs, respectively (for CTRL, the average of data obtained with 4 cell populations is shown). Cells were treated or not with IFN 24 h prior to infection with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla). Relative luminescence results for IFN-treated and -untreated conditions are shown. Two-way ANOVA on log-transformed data with Sidak's test. Bottom: Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42-depleted populations; Actin served as a loading control.


C . DDX42 silencing efficiency measured by RT - qPCR ( top ) and immunoblot ( bottom ) in parallel samples from B .

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C. DDX42 silencing efficiency measured by RT-qPCR (top) and immunoblot (bottom) in parallel samples from B.


G . Primary CD4 + T cells were electroporated with Cas9 - sgRNA RNPs using 2 non - targeting sgRNAs ( sgCTRL1 and 2 ) and 5 sgRNAs targeting DDX42 . Top : Cells were then infected with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) and relative infection efficiencies obtained with cells from 3 donors are shown . Two - way ANOVA on log - transformed data with Dunnett ' s test . Bottom : DDX42 protein levels were determined by immunoblot , Actin served as a loading control . A representative immunoblot is shown .

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G. Primary CD4+ T cells were electroporated with Cas9-sgRNA RNPs using 2 non-targeting sgRNAs (sgCTRL1 and 2) and 5 sgRNAs targeting DDX42. Top: Cells were then infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla) and relative infection efficiencies obtained with cells from 3 donors are shown. Two-way ANOVA on log-transformed data with Dunnett's test. Bottom: DDX42 protein levels were determined by immunoblot, Actin served as a loading control. A representative immunoblot is shown.


C . Heat - annealed ODN / RNA 1 - 294 complex was incubated with HIV - 1 RT and increasing concentrations of recombinant DDX42 . Reverse transcription was initiated by addition of the four dNTPs . Extension was for 1 , 5 , 20 or 60 min and samples were analysed by PAGE 8 % ( P / T : primer / template ; SSDNA : Strong - Stop DNA ) . A representative autoradiograph is shown .

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C. Heat-annealed ODN/RNA 1-294 complex was incubated with HIV-1 RT and increasing concentrations of recombinant DDX42. Reverse transcription was initiated by addition of the four dNTPs. Extension was for 1, 5, 20 or 60 min and samples were analysed by PAGE 8% (P/T: primer/template; SSDNA: Strong-Stop DNA). A representative autoradiograph is shown.


D . Quantification of 3 biological replicates performed as in C . Two - way ANOVA on log - transformed data with Dunnett ' s test .

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D. Quantification of 3 biological replicates performed as in C. Two-way ANOVA on log-transformed data with Dunnett's test.


E . PLAs were performed in MDMs infected with HIV - 1 or not ( N . I . CTRL ) , using anti - Capsid and anti - DDX42 antibodies ( nuclei stained with Hoechst ) . Images were acquired using a LSM880 Airyscan microscope . Left : representative images , scale - bar : 10 μm . Right : Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD ( n > 65 cells per condition ) .

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E. PLAs were performed in MDMs infected with HIV-1 or not (N.I. CTRL), using anti-Capsid and anti-DDX42 antibodies (nuclei stained with Hoechst). Images were acquired using a LSM880 Airyscan microscope. Left: representative images, scale-bar: 10 μm. Right: Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD (n>65 cells per condition).


K . HEK293T were co - transfected with pRPS - GFP and a Flag - Firefly - ( negative control ) or Flag - DDX42 - coding plasmid , followed by Flag immunoprecipitation and immunoblot analysis . A representative immunoblot is shown .

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K. HEK293T were co-transfected with pRPS-GFP and a Flag-Firefly- (negative control) or Flag-DDX42-coding plasmid, followed by Flag immunoprecipitation and immunoblot analysis. A representative immunoblot is shown.


( A ) Left , A549 - ACE2 cells were infected or not with SARS - CoV - 2 for 24 h prior to PLA using mouse anti - dsRNA ( J2 ) and rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) staining with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection images are shown ; scale bar : 15 μm . Right , Average punctae were quantified in 3 biological replicates with mean ± SD ( n > 75 cells per condition ) .

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(A) Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA using mouse anti-dsRNA (J2) and rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition).


Left , A549 - ACE2 cells were infected or not with SARS - CoV - 2 for 24 h prior to PLA rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) staining with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection images are shown ; scale bar : 15 μm . Right , Average punctae were quantified in 3 biological replicates with mean ± SD ( n > 75 cells per condition ) . using an anti - N

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

Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition). using an anti-N antibody


F . The following biotinylated RNAs were used to pull - down recombinant DDX42 poly ( I : C ) , CHIKV G4 , TRF2 G4 and a mutated TRF2 G4 sequence ( Mut . TRF2 G4 ) , and DDX42 was revealed by an immunoblot .

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F. The following biotinylated RNAs were used to pull-down recombinant DDX42 poly(I:C), CHIKV G4, TRF2 G4 and a mutated TRF2 G4 sequence (Mut. TRF2 G4), and DDX42 was revealed by an immunoblot.


U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining with DAPI , anti - human centromere autoantibody ( ACA ) , and antibodies for Bub1 and TOP2A . Example images are shown ( A ) . The immunofluorescence intensity ratios of centromeric TOP2A / ACA ( B ) , centromeric TOP2A / arm TOP2A ( C ) , and arm TOP2A / DNA ( D ) , were determined on around 600 ( B and C ) or 400 ( D ) chromosomes in 20 cells . ns , no statistical significance . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm .

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U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, anti-human centromere autoantibody (ACA), and antibodies for Bub1 and TOP2A. Example images are shown (A). The immunofluorescence intensity ratios of centromeric TOP2A/ACA (B), centromeric TOP2A/arm TOP2A (C), and arm TOP2A/DNA (D), were determined on around 600 (B and C) or 400 (D) chromosomes in 20 cells. ns, no statistical significance. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.


U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining with DAPI , and antibodies for Bub1 and TOP2A . Lysates of nocodazole - arrested mitotic cells were immunoblotted with the indicated antibodies ( E ) . ns , no statistical significance .

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U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, and antibodies for Bub1 and TOP2A. Lysates of nocodazole-arrested mitotic cells were immunoblotted with the indicated antibodies (E). ns, no statistical significance.


U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining . Example images are shown ( F ) .

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U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. Example images are shown (F).


U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 500 chromosomes in 15 cells ( G ) . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm .

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U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 500 chromosomes in 15 cells (G). Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.


U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . Example images of the immunofluorescence staining are shown ( H ) .

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

U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. Example images of the immunofluorescence staining are shown (H).


U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . The immunofluorescence intensity ratios of centromeric Bub1 / ACA ( I ) were determined on around 300 chromosomes in 15 cells . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm .

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

U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric Bub1/ACA (I) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.


U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . The immunofluorescence intensity ratios of centromeric TOP2A / ACA ( J ) , and centromeric TOP2A / arm TOP2A ( K ) were determined on around 300 chromosomes in 15 cells . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm .

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

U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric TOP2A/ACA (J), and centromeric TOP2A/arm TOP2A (K) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.


L U2OS cells stably expressing CB - GFP or CB - Bub1 - K - GFP were treated as in ( H ) . Example images of the immunofluorescence staining are shown .

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

L U2OS cells stably expressing CB-GFP or CB-Bub1-K-GFP were treated as in (H). Example images of the immunofluorescence staining are shown.


U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA , and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A ( A ) . Data information : Scale bars , 10 μm .

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

U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA, and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A (A). Data information: Scale bars, 10 μm.


U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA Lysates of nocodazole - arrested mitotic cells were immunoblotted ( B ) . * represents non - specific bands .

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

U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA Lysates of nocodazole-arrested mitotic cells were immunoblotted (B). * represents non-specific bands.


U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr , and subjected to immunofluorescence staining . Example images are shown ( C ) . Data information : Scale bars , 10 μm .

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

U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. Example images are shown (C). Data information: Scale bars, 10 μm.


U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr , and subjected to immunofluorescence staining . The immunofluorescence intensity ratios of centromeric H2ApT120 / ACA ( D ) , centromeric TOP2A / ACA ( E ) , and centromeric TOP2A / arm TOP2A ( F ) were determined on around 450 chromosomes in 15 cells . Means and error bars representing S . D . are shown ( D - F ; unpaired t test ) .

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

U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. The immunofluorescence intensity ratios of centromeric H2ApT120/ACA (D), centromeric TOP2A/ACA (E), and centromeric TOP2A/arm TOP2A (F) were determined on around 450 chromosomes in 15 cells. Means and error bars representing S.D. are shown (D-F; unpaired t test).


A U2OS cells were subjected to immunofluorescence staining . Example images of mitotic cells at various stages are shown . Data information : Scale bars , 10 μm .

O B-CELL_LINE 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 O O O

A U2OS cells were subjected to immunofluorescence staining. Example images of mitotic cells at various stages are shown. Data information: Scale bars, 10 μm.


B U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence staining . Example images of anaphase cells are shown . Data information : Scale bars , 10 μm .

O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD 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 U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining. Example images of anaphase cells are shown. Data information: Scale bars, 10 μm.


C U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence staining with DAPI , and antibodies for TOP2A and H2ApT120 or H3pS10 . Example images of interphase cells are shown . The cells in the top line , which is H3pS10 - positive , are in early prophase / late G2 . Data information : Scale bars , 10 μm .

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

C U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining with DAPI, and antibodies for TOP2A and H2ApT120 or H3pS10. Example images of interphase cells are shown. The cells in the top line, which is H3pS10-positive, are in early prophase/late G2. Data information: Scale bars, 10 μm.


D U2OS - LacO cells transiently expressing EGFP - LacI , EGFP - LacI - Bub1 - K ( WT or D946N ) were subjected to immunofluorescence staining . Arrows point to the transgene loci . Data information : Scale bars , 10 μm .

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

D U2OS-LacO cells transiently expressing EGFP-LacI, EGFP-LacI-Bub1-K (WT or D946N) were subjected to immunofluorescence staining. Arrows point to the transgene loci. Data information: Scale bars, 10 μm.


E , F MBP - TOP2A ( 429 - 1531 ) - 10xHis was subjected to pulldown by GST , GST - H2A , or GST - H2ApS120 , followed by immunoblotting with antibodies for MBP , GST or H2ApT120 ( E ) . The relative pulldown efficiency was determined ( F ) . Means and error bars representing S . D . from three independent experiments are shown ( unpaired t - test ) .

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

E, F MBP-TOP2A (429-1531)-10xHis was subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP, GST or H2ApT120 (E). The relative pulldown efficiency was determined (F). Means and error bars representing S.D. from three independent experiments are shown (unpaired t-test).


A , B HeLa cells and the Sgo1 - K492A mutant cells were immunostained with the indicated antibodies . Example images are shown ( A ) . The immunofluorescence intensity ratio of centromeric INCENP / ACA was determined on around 250 chromosomes in 10 cells ( B ) . Data information : Means and error bars representing S . D . are shown ; unpaired t - test ) . Scale bars , 10 μm .

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

A, B HeLa cells and the Sgo1-K492A mutant cells were immunostained with the indicated antibodies. Example images are shown (A). The immunofluorescence intensity ratio of centromeric INCENP/ACA was determined on around 250 chromosomes in 10 cells (B). Data information: Means and error bars representing S.D. are shown ; unpaired t-test). Scale bars, 10 μm.


C , D HeLa and the Sgo1 - K492A cells were exposed to nocodazole for 4 hr . Mitotic cells were cytospun onto coverslips and fixed for immunostaining . Example images are shown ( C ) . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 150 chromosomes in 10 cells ( D ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm .

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

C, D HeLa and the Sgo1-K492A cells were exposed to nocodazole for 4 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (C). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 150 chromosomes in 10 cells (D). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.