K , L . Fluorescence intensity of MB ( K ) or CD63 - eGFP ( L ) measured in axons and growth cone central domains . Each data point corresponds to one axon or a growth cone central domain . Total number of axons analyzed : 16 . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon ; GC , growth cone .

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

K,L. Fluorescence intensity of MB (K) or CD63-eGFP (L) measured in axons and growth cone central domains. Each data point corresponds to one axon or a growth cone central domain. Total number of axons analyzed: 16. n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon; GC, growth cone.


C . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : magenta for CD63 and yellow for Lamp1 or Rab5a . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP . Scale bars : 5μm .

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

C. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63 and yellow for Lamp1 or Rab5a. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP. Scale bars: 5μm.


D . Frequency ( in percentage ) of CD63 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of puncta , axons , and independent experiments analyzed : 540 puncta , 23 axons , n = 4 ( CD63 / Rab7a ) , 416 puncta , 18 axons , n = 3 ( CD63 / LysoTracker ) , 584 puncta , 17 axons , n = 2 ( CD63 / Lamp1 ) , 649 puncta , 19 axons , n = 3 ( CD63 / Rab5a ) . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP .

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

D. Frequency (in percentage) of CD63 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons, and independent experiments analyzed: 540 puncta, 23 axons, n=4 (CD63/Rab7a), 416 puncta, 18 axons, n=3 (CD63/LysoTracker), 584 puncta, 17 axons, n=2 (CD63/Lamp1), 649 puncta, 19 axons, n=3 (CD63/Rab5a). Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP.


E . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : cyan for pre - miR - 181a - 1 and yellow for Lamp1 or Rab5a . Abbreviations : Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . Scale bars : 5μm .

E. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: cyan for pre-miR-181a-1 and yellow for Lamp1 or Rab5a. Abbreviations: Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bars: 5μm.


F . Frequency ( in percentage ) of pre - miR - 181a - 1 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of puncta , axons and independent experiments analyzed : 825 puncta , 41 axons , n = 8 ( pre - miR - 181a - 1 / CD63 ) , 732 puncta , 33 axons , n = 7 ( pre - miR - 181a - 1 / Rab7a ) , 681 puncta , 29 axons , n = 5 ( pre - miR - 181a - 1 / LysoTracker ) , 501 puncta , 17 axons , n = 2 ( pre - miR - 181a - 1 / Lamp1 ) , 791 puncta , 25 axons , n = 3 ( pre - miR - 181a - 1 / Rab5a ) . Values are median with interquartile range . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP , Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 .

F. Frequency (in percentage) of pre-miR-181a-1 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 825 puncta, 41 axons, n=8 (pre-miR-181a-1/CD63), 732 puncta, 33 axons, n=7 (pre-miR-181a-1/Rab7a), 681 puncta, 29 axons, n=5 (pre-miR-181a-1/LysoTracker), 501 puncta, 17 axons, n=2 (pre-miR-181a-1/Lamp1), 791 puncta, 25 axons, n=3 (pre-miR-181a-1/Rab5a). Values are median with interquartile range. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP, Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.


G . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : magenta for CD63 , cyan for pre - miR - 181a - 1 , yellow for Rab7a and blue for LysoTracker . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . Scale bar : 5μm .

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

G. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63, cyan for pre-miR-181a-1, yellow for Rab7a and blue for LysoTracker. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bar: 5μm.


H . Frequency ( in percentage ) of pre - miR - 181a - 1 colocalization with vesicles double positive for organelle markers . Each data point corresponds to one axon . Total number of puncta , axons and independent experiments analyzed : 745 puncta , 23 axons , n = 5 ( CD63 / pre - miR - 181a - 1 / Rab7a ) , 577 puncta , 18 axons , n = 3 ( CD63 / pre - miR - 181a - 1 / LysoTracker ) . Values are median with interquartile range . Abbreviations : ns , not significant ; CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 .

H. Frequency (in percentage) of pre-miR-181a-1 colocalization with vesicles double positive for organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 745 puncta, 23 axons, n=5 (CD63/pre-miR-181a-1/Rab7a), 577 puncta, 18 axons, n=3 (CD63/pre-miR-181a-1/LysoTracker). Values are median with interquartile range. Abbreviations: ns, not significant; CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.


I . Representative 3D - STED super - resolution image . Total number of puncta , axons and growth cones analyzed : 99 puncta , 16 axons and 10 growth cones ( MB ) , 291 puncta , 36 axons and 15 growth cones ( cy3 - pre - miR - 181a - 1 ) . n = 2 ( MB ) , n = 3 ( cy3 - pre - miR - 181a - 1 ) independent experiments . Abbreviations : CD63 , CD63 - eGFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 ; MB ; molecular beacon .

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

I. Representative 3D-STED super-resolution image. Total number of puncta, axons and growth cones analyzed: 99 puncta, 16 axons and 10 growth cones (MB), 291 puncta, 36 axons and 15 growth cones (cy3-pre-miR-181a-1). n=2 (MB), n=3 (cy3-pre-miR-181a-1) independent experiments. Abbreviations: CD63, CD63-eGFP; pre-miR-181a-1, cy3-pre-miR-181a-1; MB; molecular beacon.


A . Schematic representation of the experimental protocol . B . Representative Xenopus growth cones stained with anti - Dicer and anti - Ago2 . White dashed lines delineate growth cones . Scale bars : 5µm .

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

A. Schematic representation of the experimental protocol. B. Representative Xenopus growth cones stained with anti-Dicer and anti-Ago2. White dashed lines delineate growth cones. Scale bars: 5µm.


C . Representative E13 . 5 mice brain cross - section ( dashed red line in the schematic ) comprising the optic nerve ( ON ) stained with anti - neurofilament and anti - HA antibodies to detect RGC axons and ( FLAG - HA2 ) - Dicer , respectively . Note the absence of HA signal in wild type ( WT ) mice . The white dashed line delineates the ON . Zoom of the triple stained ON ( right panel ) : Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se ( arrowheads ) . n = 3 independent experiments . Abbreviations : WT , wild type ; HA , ( FLAG - HA2 ) - Dicer ; E13 . 5 , embryonic day 13 . 5 ; ONH , optic nerve head ; ON , optic nerve . Scale bars : 30µm .

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

C. Representative E13.5 mice brain cross-section (dashed red line in the schematic) comprising the optic nerve (ON) stained with anti-neurofilament and anti-HA antibodies to detect RGC axons and (FLAG-HA2)-Dicer, respectively. Note the absence of HA signal in wild type (WT) mice. The white dashed line delineates the ON. Zoom of the triple stained ON (right panel): Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se (arrowheads). n=3 independent experiments. Abbreviations: WT, wild type; HA, (FLAG-HA2)-Dicer; E13.5, embryonic day 13.5; ONH, optic nerve head; ON, optic nerve. Scale bars: 30µm.


D . Representative P0 superior colliculus stratum griseum superficiale ( SGS ) ( sagittal sectioning of P0 brains along the dashed red line in the schematic , maximal projection of 5 µm depth ) . Note the presence of Dicer within the SGS marked with neurofilament . n = 3 independent experiments . Abbreviations : WT , wild type ; P0 , postnatal day 0 . Scale bars : 30µm .

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

D. Representative P0 superior colliculus stratum griseum superficiale (SGS) (sagittal sectioning of P0 brains along the dashed red line in the schematic, maximal projection of 5 µm depth). Note the presence of Dicer within the SGS marked with neurofilament. n=3 independent experiments. Abbreviations: WT, wild type; P0, postnatal day 0. Scale bars: 30µm.


Quantification of miRNA and pre - miRNA expression levels using the 2 - ΔCt method and U6 as normalizer , upon Sema3A ( B ) stimulation . Data are normalized to PBS control . Each data point represents a single RT - qPCR reaction . n = 3 ( B ) independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant .

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

Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Sema3A (B) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=3 (B) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.


Quantification of miRNA and pre - miRNA expression levels using the 2 - ΔCt method and U6 as normalizer , upon Slit2 ( C ) stimulation . Data are normalized to PBS control . Each data point represents a single RT - qPCR reaction . n = 4 ( C ) independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant .

Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Slit2 (C) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=4 (C) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.


Frequency ( in percentage ) of collapsed growth cones from stage 37 / 38 embryos , following a 10 min Sema3A bath application . Concentration used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 5p ) ( E ) Total number of counted growth cones is reported in the column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino .

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

Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-5p) (E) Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.


Frequency ( in percentage ) of collapsed growth cones from stage 37 / 38 embryos , following a 10 min Sema3A bath application . Concentration used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 3p ) ( F ) . Total number of counted growth cones is reported in the column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino .

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

Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-3p) (F). Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.


B . Representative images of RGC axons within the optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within the wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . C . Quantification of misprojecting axons . Each data point corresponds to one brain . Total number of brains analyzed : 45 brains ( co - MO ) , 52 brains ( miR - 181 - MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino .

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

B. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. C. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 45 brains (co-MO), 52 brains (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.


D . Schematic representation of the experimental paradigm . Concentrations used : 0 . 5μg / μL pCS2 - mCherry or pCS2 - eGFP plasmids ; 250μM miR - 181 - MO or control MO cocktail ; 50μM miR - 181 or control mimics ( top ) . Representative images of RGC axons within the optic tectum . White arrows indicate axons targeted both by MO ( green ) and miRNA mimics ( red ) ( bottom ) . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . E . Quantification of misprojecting axons . The number reported on the bars are the total number of co - electroporated axons . Note how miR - 181 mimics rescued aberrant misprojection of morphant axons in vivo . Each data point corresponds to one independent experiment . Total number of axons and brains analyzed : 96 axons , 4 brains ( co - MO + ctrl mimics ) , 101 axons , 4 brains ( miR - 181 - MO + ctrl mimics ) , 134 axons , 4 brains ( miR - 181 - MO + miR - 181 mimics ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino ; ns , not significant .

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

D. Schematic representation of the experimental paradigm. Concentrations used: 0.5μg/μL pCS2-mCherry or pCS2-eGFP plasmids; 250μM miR-181-MO or control MO cocktail; 50μM miR-181 or control mimics (top). Representative images of RGC axons within the optic tectum. White arrows indicate axons targeted both by MO (green) and miRNA mimics (red) (bottom). Abbreviations: co-MO, control morpholino. Scale bars: 20µm. E. Quantification of misprojecting axons. The number reported on the bars are the total number of co-electroporated axons. Note how miR-181 mimics rescued aberrant misprojection of morphant axons in vivo. Each data point corresponds to one independent experiment. Total number of axons and brains analyzed: 96 axons, 4 brains (co-MO+ctrl mimics), 101 axons, 4 brains (miR-181-MO + ctrl mimics), 134 axons, 4 brains (miR-181-MO+miR-181 mimics). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino; ns, not significant.


F . Representative images of RGC axons within the optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within the wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . G . Quantification of misprojecting axons . Each data point corresponds to one brain . Total number of brains analyzed : 21 brains ( co - MO ) , 31 brains ( miR - 181 - MO ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino .

F. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. G. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 21 brains (co-MO), 31 brains (miR-181-MO). n=3 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.


I . Frequency ( in percentage ) of the amount of time embryos spent on black background . Each data point corresponds to one embryo . Total number of embryos analyzed : 43 embryos ( co - MO ) , 45 embryos ( miR - 181 - MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino .

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

I. Frequency (in percentage) of the amount of time embryos spent on black background. Each data point corresponds to one embryo. Total number of embryos analyzed: 43 embryos (co-MO), 45 embryos (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; 100µM cycloheximide Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . ( B ) 200ng / mL Sema3A was bath applied with or without 100µM cycloheximide ( CHX , a translational blocker ) . Mutating TUBB3 3 ' UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 100µM cycloheximide Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). (B) 200ng/mL Sema3A was bath applied with or without 100µM cycloheximide (CHX, a translational blocker). Mutating TUBB3 3'UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . , isolated axons ( C ) were used for FRAP analysis .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). , isolated axons (C) were used for FRAP analysis.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; 2µM co - MO or MOs - 3p . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . isolated transfected axons ( D ) were used for FRAP analysis .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 2µM co-MO or MOs-3p. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). isolated transfected axons (D) were used for FRAP analysis.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . In vivo ( E ) , whole embryos with exposed brain were used . The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution . ( E ) The red area on the brain schematic indicate Sema3A expressing territories . Numbers of axons analyzed are reported between brackets .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). In vivo (E), whole embryos with exposed brain were used. The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution. (E) The red area on the brain schematic indicate Sema3A expressing territories. Numbers of axons analyzed are reported between brackets.


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP analysis . APP , Amyloid beta precursor

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. APP, Amyloid beta precursor protein


Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP analysis . THBS1 , Thrombospondin

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. THBS1, Thrombospondin 1


H , I . Representative growth cones depicting Venus fluorescence intensity as a heatmap . Dashed white lines delineate the growth cones . Abbreviations : wt , wild type ; mut , miR - 181 - 5p responsive elements mutated ; CHX , cycloheximide ; TUBB3 , tubulin beta 3 class III . Scale bars : 10µm .

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

H,I. Representative growth cones depicting Venus fluorescence intensity as a heatmap. Dashed white lines delineate the growth cones. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III. Scale bars: 10µm.


B . Schematic representation of the experimental paradigm . Representative images of local translation events of TUBB3 in the growth cone . Dashed white line delineates the growth cone . Each white arrow points to a puro - PLA puncta . Abbreviations : TUBB3 , tubulin beta 3 class III ; Puro , puromycin . Scale bars : 5 μm . Quantification of number of puncta per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 30 ( PBS ) , 36 ( Sema3A ) . n = 2 independent experiments . Values are median with interquartile range .

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

B. Schematic representation of the experimental paradigm. Representative images of local translation events of TUBB3 in the growth cone. Dashed white line delineates the growth cone. Each white arrow points to a puro-PLA puncta. Abbreviations: TUBB3, tubulin beta 3 class III; Puro, puromycin. Scale bars: 5 μm. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 30 (PBS), 36 (Sema3A). n= 2 independent experiments. Values are median with interquartile range.


D . Schematic representation of the experimental paradigm . Concentrations used : 2 μM co - MO ; 2 μM MOs - 3p ; 200ng / mL Sema3A . Representative images of local translation events of TUBB3 in the growth cone of transfected axons . Dashed white line delineates the growth cone . White arrows represent number of puro - PLA puncta per growth cone . Abbreviations : co - MO , control morpholino . Scale bars : 5 μm . E . Quantification of number of puncta per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 32 ( co - MO + PBS ) , 45 ( co - MO + Sema3A ) , 27 ( MOs - 3p + Sema3A ) . n = 2 independent experiments . Values are median with interquartile range . Abbreviations : co - MO , control morpholino ; ns , not significant .

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

D. Schematic representation of the experimental paradigm. Concentrations used: 2 μM co-MO; 2 μM MOs-3p; 200ng/mL Sema3A. Representative images of local translation events of TUBB3 in the growth cone of transfected axons. Dashed white line delineates the growth cone. White arrows represent number of puro-PLA puncta per growth cone. Abbreviations: co-MO, control morpholino. Scale bars: 5 μm. E. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 32 (co-MO + PBS), 45 (co-MO + Sema3A), 27 (MOs-3p + Sema3A). n=2 independent experiments. Values are median with interquartile range. Abbreviations: co-MO, control morpholino; ns, not significant.


G . Percentage of collapsed growth cones in axons transfected with co - MO , MOs - 3p + co - MO and MOs - 3p + MOs - TUBB3 . Each data point corresponds to one independent experiment . Total number of growth cones counted : 152 ( co - MO ) , 155 ( MOs - 3p + coMO ) , 179 ( MOs - 3p + MOs - TUBB3 ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino ; TUBB3 , tubulin beta 3 class III .

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

G. Percentage of collapsed growth cones in axons transfected with co-MO, MOs-3p + co-MO and MOs-3p + MOs-TUBB3. Each data point corresponds to one independent experiment. Total number of growth cones counted: 152 (co-MO), 155 (MOs-3p + coMO), 179 (MOs-3p + MOs-TUBB3). n= 3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; TUBB3, tubulin beta 3 class III.


B . Blood stage growth rate of mutated parasite lines . A single parasite was injected i . v . into a mouse and the growth rate was calculated from parasitemia at day 6 - 9 for each mouse ( black dots ) . Bars represent the mean of 4 - 7 mice . Significance for ( B ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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

B. Blood stage growth rate of mutated parasite lines. A single parasite was injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9 for each mouse (black dots). Bars represent the mean of 4-7 mice. Significance for (B) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


C . Infected midgut showing oocysts stained with mercurochrome ; scale bar , 100 μm . The graph shows oocyst numbers per infected mosquito . Numbers of mosquitoes that were analyzed are indicated above the graph . Data derived from at least two independent cage feeds originating from independently infected mice . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance for ( C ) determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

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

C. Infected midgut showing oocysts stained with mercurochrome; scale bar, 100 μm. The graph shows oocyst numbers per infected mosquito. Numbers of mosquitoes that were analyzed are indicated above the graph. Data derived from at least two independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (C) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


D . Ratio of salivary gland to midgut sporozoites of the indicated parasite lines . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Significance for , ( D ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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

D. Ratio of salivary gland to midgut sporozoites of the indicated parasite lines. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for , (D) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


E . Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging . Arrow indicates direction of a motile sporozoite . The graph shows the fraction of motile hemolymph sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Significance for ( E ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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

E. Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging. Arrow indicates direction of a motile sporozoite. The graph shows the fraction of motile hemolymph sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for (E) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


F . qRT - PCR analysis of schizonts and oocysts of the PbMyoA 3 ' dhfs parasite line . Individual data points correspond to the mean of three technical replicates .

O O O O O O O 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. qRT-PCR analysis of schizonts and oocysts of the PbMyoA 3'dhfs parasite line. Individual data points correspond to the mean of three technical replicates.


C . Blood stage growth rate of clonal parasite lines . A single parasite or 100 iRBCs were injected i . v . into a mouse and the growth rate was calculated from parasitemia at day 6 - 9 . Black dots : individual mice ; bars : mean . Statistical analysis : ( C ) One - way analysis of variance with Tukeys multiple comparison test .

C. Blood stage growth rate of clonal parasite lines. A single parasite or 100 iRBCs were injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9. Black dots: individual mice; bars: mean. Statistical analysis: (C) One-way analysis of variance with Tukeys multiple comparison test.


D . Proportion of motile ookinetes . Black dots : Individual movie ; bars : mean . Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment . Statistical analysis : ( D ) Kruskal - Wallis test with ( D ) Dunn ' s multiple comparisons

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Proportion of motile ookinetes. Black dots: Individual movie; bars: mean. Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment. Statistical analysis: (D) Kruskal-Wallis test with (D) Dunn's multiple comparisons test


E . Speed of ookinetes of the different lines . Numbers of analysed parasites are shown above the graph . Data pooled from three independent experiments ( biological replicates ) . Violin plots show median ( line ) and quartiles ( dashed lines ) .

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

E. Speed of ookinetes of the different lines. Numbers of analysed parasites are shown above the graph. Data pooled from three independent experiments (biological replicates). Violin plots show median (line) and quartiles (dashed lines).


F . Number of oocysts per infected mosquito . Numbers of analyzed mosquitoes are shown above the graph . Data pooled from at least three independent cage feeds originating from independently infected mice . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Statistical analysis : ( F ) Kruskal - Wallis test with ( F ) Bonferroni ' s Multiple Comparison test .

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

F. Number of oocysts per infected mosquito. Numbers of analyzed mosquitoes are shown above the graph. Data pooled from at least three independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Statistical analysis: (F) Kruskal-Wallis test with (F) Bonferroni's Multiple Comparison test.


A . Ratio of hemolymph to midgut sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . B . Ratio of salivary gland to midgut sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Statistical analysis : ( A ) and ( B ) One - way analysis of variance with Tukeys Multiple Comparison test .

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

A. Ratio of hemolymph to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. B. Ratio of salivary gland to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Statistical analysis: (A) and (B) One-way analysis of variance with Tukeys Multiple Comparison test.


C . Blood stage infection of mice after transmission by mosquito bite . Shown is the mean ± standard deviation . D . Blood stage infection of mice after i . v . injection of 1000 sporozoites .

O O O O O O O O O O O O O O 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. Blood stage infection of mice after transmission by mosquito bite. Shown is the mean ± standard deviation. D. Blood stage infection of mice after i.v. injection of 1000 sporozoites.


AFraction ( left ) and speed ( right ) of motile hemolymph sporozoites on glass . Dots correspond to individual experiments and bars indicate the mean . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance for ( A left ) and ( B left ) determined by One - way analysis of variance with Tukeys Multiple Comparison test . Significance for ( A right ) and ( B right ) determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

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

AFraction (left) and speed (right) of motile hemolymph sporozoites on glass . Dots correspond to individual experiments and bars indicate the mean. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (A left) and (B left) determined by One-way analysis of variance with Tukeys Multiple Comparison test. Significance for (A right) and (B right) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min . Scale bar , 10 μm . D . Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min . Scale bar , 10 μm .

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

C. Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min. Scale bar, 10 μm. D. Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min. Scale bar, 10 μm.


A . Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap . The bead is placed at the apical end of the sporozoite . The graph shows the fraction of sporozoites that pulled the bead out of the optical trap . Black dots : individual experiments . Bars : mean . 73 - 131 sporozoites were probed for each line . Significance determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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

A. Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap. The bead is placed at the apical end of the sporozoite. The graph shows the fraction of sporozoites that pulled the bead out of the optical trap. Black dots: individual experiments. Bars: mean. 73-131 sporozoites were probed for each line. Significance determined by One-way analysis of variance with Tukeys Multiple Comparison test.


B . Scheme showing a sporozoite moving through a polymeric network in 3D . The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

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

B. Scheme showing a sporozoite moving through a polymeric network in 3D. The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 moving sporozoites observed over a period of 3 min . Scale bar , 10 μm .

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

C. Selected trajectories of 20 moving sporozoites observed over a period of 3 min. Scale bar, 10 μm.


( A ) Representative transmission electron microscope ( TEM ) image showing the presence of EVs in the CSF in two independent experiments .

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

(A) Representative transmission electron microscope (TEM) image showing the presence of EVs in the CSF in two independent experiments.


( B ) NanoSightquantification of the amount of particles in the CSF 0 , 1 , 2 , 4 and 6 hours after i . p . LPS injection ( n = 3 - 5 ) .

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

(B) NanoSightquantification of the amount of particles in the CSF 0, 1, 2, 4 and 6 hours after i.p. LPS injection (n=3-5).


( C ) Size distribution of the EVsin vivo in the CSF before ( black ; n = 5 ) and 6 h after ( grey ; n = 3 ) LPS treatment determined by NanoSight analysis .

(C) Size distribution of the EVsin vivo in the CSF before (black; n=5) and 6 h after (grey; n=3) LPS treatment determined by NanoSight analysis.


( D - G ) Quantitative real - time polymerase chain reaction analysis of miR - 1a ( D ) , miR - 9 ( E ) , miR - 146a ( F ) , and miR - 155 ( G ) ( n = 4 ) . RNA was isolated from pooled CSF ( 50 µl ) from different mice ( n = 3 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Quantitative real-time polymerase chain reaction analysis of miR-1a (D), miR-9 (E), miR-146a (F), and miR-155 (G) (n=4). RNA was isolated from pooled CSF (50 µl) from different mice (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A - B ) In vitro quantification ( A ) and size distribution ( B ) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence ( black ) or presence ( grey ) of LPS ( n = 5 ) determined by NanoSight analysis .

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(A-B) In vitro quantification (A) and size distribution (B) of EVs isolated from conditioned medium of primary CPE cells grown in transwell system after 12 h in the absence (black) or presence (grey) of LPS (n=5) determined by NanoSight analysis.


( C - E ) TaqMan qPCR assay for the quantification of miR - 9 ( C ) , miR - 146a ( D ) and miR - 155 ( E ) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS ( n = 3 ) .

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(C-E) TaqMan qPCR assay for the quantification of miR-9 (C), miR-146a (D) and miR-155 (E) on the exosomal pellet isolated from conditioned medium of primary CPE cells grown in a transwell system and stimulated for 12 h with LPS (n=3).


( F - I ) Quantification of the miRNAs miR - 1a ( F ) , miR - 9 ( G ) , miR - 146a ( H ) and miR - 155 ( I ) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation ( n = 3 ) . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(F-I) Quantification of the miRNAs miR-1a (F), miR-9 (G), miR-146a (H) and miR-155 (I) by TaqMan qPCR assay from primary CPE cells grown in a transwell system without or with LPS stimulation (n=3). Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) In vitro quantification of EVs isolated from conditioned medium of LPS - stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 ( n = 3 ) .

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(A) In vitro quantification of EVs isolated from conditioned medium of LPS-stimulated primary CPE cells grown in a transwell system in the absence or presence of the exosome inhibitor GW4869 (n=3).


( B - D ) TaqMan assay quantification of the miRNAs miR - 9 ( B ) , miR - 146a ( C ) and miR - 155 ( D ) in supernatant of LPS - stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion ( n = 3 ) . miR - 1a levels were below detection limit .

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(B-D) TaqMan assay quantification of the miRNAs miR-9 (B), miR-146a (C) and miR-155 (D) in supernatant of LPS-stimulated primary CPE cells grown in a transwell system and either left untreated or pretreated with GW4869 to inhibit exosome secretion (n=3). miR-1a levels were below detection limit.


( E - H ) TaqMan assay quantification of the miRNAs miR - 1a ( E ) , miR - 9 ( F ) , miR - 146a ( G ) and miR - 155 ( H ) in cell lysate of LPS - stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion ( n = 3 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 .

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(E-H) TaqMan assay quantification of the miRNAs miR-1a (E), miR-9 (F), miR-146a (G) and miR-155 (H) in cell lysate of LPS-stimulated primary CPE cells grown in a transwell system left untreated or treated with GW4869 to inhibit exosome secretion (n=3). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01.


( A ) Representative confocal images of CD63 , RAB5 and ANXA2 ( red ) in the choroid plexus ( CP ) 0 , 4 and 8 h after LPS treatment . Hoechst ( blue ) was used to stain the nucleus . The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image . Scalebar = 100 µm .

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(A) Representative confocal images of CD63, RAB5 and ANXA2 (red) in the choroid plexus (CP) 0, 4 and 8 h after LPS treatment. Hoechst (blue) was used to stain the nucleus. The dotted line indicates the ependymal cells that line the ventricle and the square boxes indicate the zoomed insert images displayed at the right corner of each image. Scalebar = 100 µm.


( B - C ) Representative TEM images showing the presence of MVBs in the CPE cells before ( B ) and 6 hours after ( C ) LPS administration in vivo . White arrow heads point to exosomes present in MVBs ( n = 3 ) . Scale bar = 9 µm .

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(B-C) Representative TEM images showing the presence of MVBs in the CPE cells before (B) and 6 hours after (C) LPS administration in vivo. White arrow heads point to exosomes present in MVBs (n=3). Scale bar = 9 µm.


( D - F ) Quantification of amount of MVBs per cell section ( D ) , amount of exosomes per MVB ( E ) , and amount of exosomes per cell section ( F ) , based on TEM analysis .

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(D-F) Quantification of amount of MVBs per cell section (D), amount of exosomes per MVB (E), and amount of exosomes per cell section (F), based on TEM analysis.


( G - J ) Quantitative real - time polymerase chain reaction ( qPCR ) analysis of miR - 1a ( G ) , miR - 9 ( H ) , miR - 146a ( I ) , and miR - 155 ( J ) . Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay ( 0 h , n = 4 ; 1 h , n = 5 ; 6 h , n = 5 ; 24 h , n = 3 ) .

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(G-J) Quantitative real-time polymerase chain reaction (qPCR) analysis of miR-1a (G), miR-9 (H), miR-146a (I), and miR-155 (J). Data are presented as relative expression normalized with housekeeping miRs by TaqMan qPCR assay (0 h, n=4; 1 h, n=5; 6 h, n=5; 24 h, n=3).


( K ) NanoSight analysis of CSF isolated from LPS - injected mice followed by icv injection of vehicle or GW4869 , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 8 ) .

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(K) NanoSight analysis of CSF isolated from LPS-injected mice followed by icv injection of vehicle or GW4869, a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 8).


( L ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle ( black ) or GW4869 ( grey ) , a neutral sphingomyelinase inhibitor that inhibits exosome secretion ( n = 4 ) .

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(L) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of mice injected with LPS and then icv injected with vehicle (black) or GW4869 (grey), a neutral sphingomyelinase inhibitor that inhibits exosome secretion (n = 4).


( M ) NanoSight analysis of supernatant of choroid plexus explants from PBS - or LPS - injected mice ( n = 6 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

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(M) NanoSight analysis of supernatant of choroid plexus explants from PBS- or LPS-injected mice (n = 6). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A - B ) Representative confocal images of choroid plexus ( CP ) on brain sections from naive mice and four hours after LPS injection ( n = 3 ) . Brain sections were stained for TLR4 ( A ) and TNFR1 ( B ) ( red ) , pan - cytokeratine ( panCK ; green ) and the nuclei were stained with Hoechst ( blue ) . The ependymal cells aligning the ventricles are marked with a dotted line . Scalebar = 100 µm .

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(A-B) Representative confocal images of choroid plexus (CP) on brain sections from naive mice and four hours after LPS injection (n=3). Brain sections were stained for TLR4 (A) and TNFR1 (B) (red), pan-cytokeratine (panCK; green) and the nuclei were stained with Hoechst (blue). The ependymal cells aligning the ventricles are marked with a dotted line. Scalebar = 100 µm.


( C ) NanoSight analysis of EVs in the CSF in sham - operated ( black ) relative to CLP - treated ( grey ) mice 10 hours after surgery .

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(C) NanoSight analysis of EVs in the CSF in sham-operated (black) relative to CLP-treated (grey) mice 10 hours after surgery.


( D ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery . ( E ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of sham - operated mice ( black ) and mice subjected to CLP ( grey ) 10 hours after surgery .

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(D) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery. (E) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of sham-operated mice (black) and mice subjected to CLP (grey) 10 hours after surgery.


( F ) NanoSight analysis of EVs in CSF of control ( black ) and TNF - injected ( 25 µg / 20 g ; grey ) mice 6 hours after TNF challenge .

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(F) NanoSight analysis of EVs in CSF of control (black) and TNF-injected (25 µg/20 g; grey) mice 6 hours after TNF challenge.


( G ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the choroid plexus of control mice ( black ) and in mice injected with TNF ( grey ) 6 hours after injection . ( H ) qPCR analysis of the expression of miR - 1a , miR - 9 , miR - 146a and miR - 155 in the CSF of control mice ( black ) and on mice 6 hours after TNF injection ( grey ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Scalebar 100 µm . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 ; * * , 0 . 001 ≤ P < 0 . 01 ; * * * , 0 . 0001 ≤ P < 0 . 001 .

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(G) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the choroid plexus of control mice (black) and in mice injected with TNF (grey) 6 hours after injection. (H) qPCR analysis of the expression of miR-1a, miR-9, miR-146a and miR-155 in the CSF of control mice (black) and on mice 6 hours after TNF injection (grey). Data are displayed as mean ± SEM and analyzed by Student's t-test. Scalebar 100 µm. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, 0.0001 ≤ P < 0.001.


( A ) Representative confocal image of brain parenchyma , four hours after intracerebroventricular injection of PKH26 labelled EVs ( red ) . Astrocytes are stained with GFAP ( white ) , nuclei with Hoechst ( blue ) , and pan - cytokeratin ( panCK ; green ) was used to label the choroid plexus . The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer .

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(A) Representative confocal image of brain parenchyma, four hours after intracerebroventricular injection of PKH26 labelled EVs (red). Astrocytes are stained with GFAP (white), nuclei with Hoechst (blue), and pan-cytokeratin (panCK; green) was used to label the choroid plexus. The dotted line shows the ventricular border and the white arrow heads point to EVs that crossed the ependymal cell layer.


( B - C ) Representative confocal images showing the uptake of double - labelled EVs ( Ribogreen ; green , PKH26 , red ) by astrocytes stained with GFAP ( white ) ( B ) and microglia cells stained with IBA1 ( white ) ( C ) incubated on mixed cortical cultures . Boxed areas are shown as zoomed images on right . Cell nuclei are stained with Hoechst ( blue ) . The in vivo and in vitro uptake experiments were performed 3 and 2 times ( n = 3 ) , respectively . Scalebar 100 = µm .

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(B-C) Representative confocal images showing the uptake of double-labelled EVs (Ribogreen; green, PKH26, red) by astrocytes stained with GFAP (white) (B) and microglia cells stained with IBA1 (white) (C) incubated on mixed cortical cultures. Boxed areas are shown as zoomed images on right. Cell nuclei are stained with Hoechst (blue). The in vivo and in vitro uptake experiments were performed 3 and 2 times (n=3), respectively. Scalebar 100 = µm.


( A - B ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice ( A ) and in brain tissue ( B ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

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(A-B) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 in mixed cortical cultures incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice (A) and in brain tissue (B) before (black) and 8 h after LPS injection (grey) (n=3).


( C ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα by qPCR in mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from CSF from untreated ( black ) or LPS treated ( grey ) mice and in brain tissue ( right ; in vivo ) before ( black ) and 8 h after LPS injection ( grey ) ( n = 3 ) .

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(C) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα by qPCR in mixed cortical cultures (left; in vitro) incubated with EVs isolated from CSF from untreated (black) or LPS treated (grey) mice and in brain tissue (right; in vivo) before (black) and 8 h after LPS injection (grey) (n=3).


( D ) Cytokine analysis ( IL - 6 , IL - 1β and TNF ) of supernatant of mixed cortical cultures ( left ; in vitro ) incubated with EVs isolated from untreated ( black ; n = 3 ) or LPS treated ( grey ; n = 3 ) mice and of CSF ( right ; in vivo ) from untreated ( black ; n = 3 ) and LPS treated mice ( grey ; n = 6 ) .

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(D) Cytokine analysis (IL-6, IL-1β and TNF) of supernatant of mixed cortical cultures (left; in vitro) incubated with EVs isolated from untreated (black; n=3) or LPS treated (grey; n=3) mice and of CSF (right; in vivo) from untreated (black; n=3) and LPS treated mice (grey; n=6).


( E - F ) qPCR gene expression analysis of inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( E ) and miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( F ) in mixed cortical cultures incubated with CD63 - depleted EVs isolated from untreated mice ( black ) and mice treated with LPS for 6 h ( grey ) ( n = 3 ) .

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(E-F) qPCR gene expression analysis of inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (E) and miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (F) in mixed cortical cultures incubated with CD63-depleted EVs isolated from untreated mice (black) and mice treated with LPS for 6 h (grey) (n=3).


( G - H ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( G ) and inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( H ) in brain tissue from LPS - injected mice icv injected with vehicle ( black ) or GW4869 ( grey ; n = 7 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 .

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(G-H) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (G) and inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (H) in brain tissue from LPS-injected mice icv injected with vehicle (black) or GW4869 (grey; n=7). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05.


Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines : MCF7 parental or doxorubicin - resistant MCF7 / ADR ( ADR , 0 . 5μg / ml ) and irradiation - resistant MCF7 / IR ( IR , 10Gy ) , MDA - MB - 231 parental or doxorubicin - resistant MDA - MB - 231 / ADR ( ADR , 0 . 5μg / ml ) or cisplatin - resistant MDA - MB - 231 / DDP ( 1μg / ml ) , non - small lung cancer cell lines : A549 parental or cisplatin - resistant A549 / DDP ( 1μg / ml ) .

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Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines: MCF7 parental or doxorubicin-resistant MCF7/ADR (ADR, 0.5μg/ml) and irradiation-resistant MCF7/IR (IR, 10Gy), MDA-MB-231 parental or doxorubicin-resistant MDA-MB-231/ADR (ADR, 0.5μg/ml) or cisplatin-resistant MDA-MB-231/DDP (1μg/ml), non-small lung cancer cell lines: A549 parental or cisplatin-resistant A549/DDP (1μg/ml).


MCF7 , MCF7 / ADR and MCF7 / IR , MDA - MB - 231 and MDA - MB - 231 / ADR cells were harvested and mRNA levels of BRD7 were determined by real - time PCR ( n = 3 ) . Values are mean ± SEM and Error bars indicate SEM .

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

MCF7, MCF7/ADR and MCF7/IR, MDA-MB-231 and MDA-MB-231/ADR cells were harvested and mRNA levels of BRD7 were determined by real-time PCR (n=3). Values are mean ± SEM and Error bars indicate SEM.


Western blot analysis of BRD7 protein levels in MDA - MB - 468 and MDA - MB - 231 cell after treatment with ADR ( 5 μM ) or camptothecin ( CPT ) ( 1 μM ) for different intervals ( n = 3 ) .

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

Western blot analysis of BRD7 protein levels in MDA-MB-468 and MDA-MB-231 cell after treatment with ADR (5 μM) or camptothecin (CPT) (1 μM) for different intervals (n=3).


Representative images of endogenous BRD7 ( green ) and γH2AX foci ( red ) in paraformaldehyde - fixed MDA - MB - 468 cells after treatment with CPT ( 1 μM ) for different intervals . Visualized by immunofluorescence using anti - BRD7 and Alexa Fluor 555 anti - γH2AX antibodies . DNA staining with DAPI ; Scale bars , 2 μm .

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

Representative images of endogenous BRD7 (green) and γH2AX foci (red) in paraformaldehyde-fixed MDA-MB-468 cells after treatment with CPT (1 μM) for different intervals. Visualized by immunofluorescence using anti-BRD7 and Alexa Fluor 555 anti-γH2AX antibodies. DNA staining with DAPI; Scale bars, 2 μm.


Quantification of average fluorescence intensity of BRD7 of cells in ( E ) . Error bars indicate SEM ; n > 100 .

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

Quantification of average fluorescence intensity of BRD7 of cells in (E). Error bars indicate SEM; n>100.


Silver staining of the BRD7 complex separated by SDS - PAGE . HEK293T cells stably expressing SFB - tagged BRD7 were used for tandem affinity purification ( TAP ) of protein complexes . BRD7 - interacting proteins , including PARP1 and PIK3R2 , are indicated .

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

Silver staining of the BRD7 complex separated by SDS-PAGE. HEK293T cells stably expressing SFB-tagged BRD7 were used for tandem affinity purification (TAP) of protein complexes. BRD7-interacting proteins, including PARP1 and PIK3R2, are indicated.


HEK293T cells transiently transfected with Flag - PARP1 and Myc - BRD7 for 24 h were lysed with RIPA buffer . Followed by immunoprecipitation ( IP ) using antibodies to Myc ( C ) conjugated to agarose followed by Western blot with the indicated antibodies ( n = 3 ) .

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

HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to Myc (C) conjugated to agarose followed by Western blot with the indicated antibodies (n=3).


HEK293T cells transiently transfected with Flag - PARP1 and Myc - BRD7 for 24 h were lysed with RIPA buffer . Followed by immunoprecipitation ( IP ) using antibodies to Flag ( D ) conjugated to agarose followed by Western blot with the indicated antibodies ( n = 3 ) .

HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to Flag (D) conjugated to agarose followed by Western blot with the indicated antibodies (n=3).


HeLa and MDA - MB - 231 cells were lysed with RIPA buffer , and lysates were subjected to immunoprecipitation using either anti - IgG , or BRD7 or PARP1 antibodies , and analysed by Western blot ( n = 3 ) .

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

HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG, or BRD7 or PARP1 antibodies, and analysed by Western blot (n=3).


MDA - MB - 231 cells were treated first with Olaparib ( 10 μM ) for 6 h and lysed with RIPA buffer , and lysates were subjected to immunoprecipitation using either anti - IgG or PARP1 antibodies , and analysed by Western blot ( n = 3 ) .

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

MDA-MB-231 cells were treated first with Olaparib (10 μM) for 6 h and lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG or PARP1 antibodies, and analysed by Western blot (n=3).


Association of endogenous BRD7 with PARP1 in HeLa cells was performed by co - immunoprecipitation using anti - BRD7 or anti - PARP1 antibody . HeLa cell were treated with CPT ( 1 μM , 1 h ) , followed by IP using indicated antibodies , and Western blot was performed . γH2AX was used as a marker of DNA damage induced by CPT ( n = 3 ) .

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

Association of endogenous BRD7 with PARP1 in HeLa cells was performed by co-immunoprecipitation using anti-BRD7 or anti-PARP1 antibody. HeLa cell were treated with CPT (1 μM, 1 h), followed by IP using indicated antibodies, and Western blot was performed. γH2AX was used as a marker of DNA damage induced by CPT (n=3).


HeLa cells were untreated or treated with CPT ( 1 μM ) for 1 h followed by lysing with RIPA buffer and lysates were then immunoprecipitated using anti - IgG or anti - PAR antibodies and immunoblotted with the indicated antibodies ( n = 3 ) .

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

HeLa cells were untreated or treated with CPT (1 μM) for 1 h followed by lysing with RIPA buffer and lysates were then immunoprecipitated using anti-IgG or anti-PAR antibodies and immunoblotted with the indicated antibodies (n=3).


HeLa cells were untreated or treated with CPT ( 1 μM ) for 1 h and cellular lysates were immunoprecipitated using anti - IgG or anti - BRD7 antibodies and immunoblotted were performed using the indicated antibodies ( n = 3 ) .

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

HeLa cells were untreated or treated with CPT (1 μM) for 1 h and cellular lysates were immunoprecipitated using anti-IgG or anti-BRD7 antibodies and immunoblotted were performed using the indicated antibodies (n=3).


HeLa and 293T cells transfected with Myc - BRD7 plasmid for 24 h were lysed with RIPA buffer . Lysates were then immunoprecipitated using anti - Myc agarose and immunoblotted using the indicated antibodies . Ribosylation levels of exogenous BRD7 were detected using anti - PAR antibody ( n = 3 ) .

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

HeLa and 293T cells transfected with Myc-BRD7 plasmid for 24 h were lysed with RIPA buffer. Lysates were then immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies. Ribosylation levels of exogenous BRD7 were detected using anti-PAR antibody (n=3).


HeLa cells transfected with Myc - BRD7 plasmid . After 24 hours , cells were treated with either CPT ( 1 μM ) or ADR ( 5 μM ) combined with MG132 ( 10 μM ) for indicated times . Cellular lysates were immunoprecipitated using anti - Myc agarose and immunoblotted using the indicated antibodies ( n = 3 ) .

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

HeLa cells transfected with Myc-BRD7 plasmid. After 24 hours, cells were treated with either CPT (1 μM) or ADR (5 μM) combined with MG132 (10 μM) for indicated times. Cellular lysates were immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies (n=3).


HeLa PARP1 wild - type and PARP1 knockout cells were transfected with Myc - BRD7 for 24 h , lysates were subjected to immunoprecipitation using anti - Myc agarose and analysed by Western blot ( n = 3 ) .

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

HeLa PARP1 wild-type and PARP1 knockout cells were transfected with Myc-BRD7 for 24 h, lysates were subjected to immunoprecipitation using anti-Myc agarose and analysed by Western blot (n=3).


HeLa were transfected with BRD7 wild - type and various BRD7 mutant plasmids for 24 h , lysed with RIPA , followed by anti - Myc IP and Western blot with indicated antibody ( n = 3 ) .

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

HeLa were transfected with BRD7 wild-type and various BRD7 mutant plasmids for 24 h, lysed with RIPA, followed by anti-Myc IP and Western blot with indicated antibody (n=3).


Ribosylation of BRD7 by PARP1 in vitro . Recombinant BRD7 was subjected to in vitro ribosylation either in absence or presence of biotin - labeled NAD + . Recombinant proteins were detected by indicated antibodies , and ribosylated proteins were determined with anti - biotin antibody ( n = 3 ) .

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

Ribosylation of BRD7 by PARP1 in vitro. Recombinant BRD7 was subjected to in vitro ribosylation either in absence or presence of biotin-labeled NAD+. Recombinant proteins were detected by indicated antibodies, and ribosylated proteins were determined with anti-biotin antibody (n=3).


PAR - binding motif of BRD7 is required for its ribosylation by PARP1 . Recombinant Myc - BRD7 - WT and Myc - BRD7 - mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated ( n = 3 ) .

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

PAR-binding motif of BRD7 is required for its ribosylation by PARP1. Recombinant Myc-BRD7-WT and Myc-BRD7-mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated (n=3).


HeLa cells were transfected with Flag - PARP1 and Myc - BRD7 for 24 h , then treated with MG132 ( 10 μM ) for additional 4 h , and proteins were detected by Western blot with indicated antibodies ( n = 3 ) .

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

HeLa cells were transfected with Flag-PARP1 and Myc-BRD7 for 24 h, then treated with MG132 (10 μM) for additional 4 h, and proteins were detected by Western blot with indicated antibodies (n=3).


HeLa cells were transfected with either scrambled or PARP1 siRNAs for 48 h , protein levels were detected by Western blot with the indicated antibodies ( n = 3 ) .

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

HeLa cells were transfected with either scrambled or PARP1 siRNAs for 48 h, protein levels were detected by Western blot with the indicated antibodies (n=3).


Expression of BRD7 , p21 and PARP1 measured in PARP1 wild - type and knockout HeLa cells using PARP1 sgRNA by Western blot ( n = 3 ) .

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Expression of BRD7, p21 and PARP1 measured in PARP1 wild-type and knockout HeLa cells using PARP1 sgRNA by Western blot (n=3).


MDA - MB - 231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h , cell lysates were analysed by Western blot ( n = 3 ) .

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MDA-MB-231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h, cell lysates were analysed by Western blot (n=3).


MDA - MB - 231 cell were treated with 10 μM of PDD0017273 for indicated times , cell lysates were analysed by Western blot ( n = 3 ) .

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MDA-MB-231 cell were treated with 10 μM of PDD0017273 for indicated times, cell lysates were analysed by Western blot (n=3).


Wild - type and PARP1 knockout HeLa cells were incubated with 10 μg / ml cycloheximide ( CHX ) for the indicated times . Lysates were harvested and analysed by Western blot . Quantification of BRD7 protein are shown in F ( n = 3 ) and results represent mean ± SEM . Error bars indicate SEM . Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells , respectively .

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

Wild-type and PARP1 knockout HeLa cells were incubated with 10 μg/ml cycloheximide (CHX) for the indicated times. Lysates were harvested and analysed by Western blot. Quantification of BRD7 protein are shown in F (n=3) and results represent mean ± SEM. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells, respectively.


PARP1 wild - type and knockout HeLa cells were transfected with HA - ubiquitin for 24 h , and MG132 ( 10 μM ) was added for an additional 4 h , cells were lysed with RIPA buffer ; followed by anti - BRD7 IP and analysed by Western blot with the indicated antibodies ( n = 3 ) .

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

PARP1 wild-type and knockout HeLa cells were transfected with HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h, cells were lysed with RIPA buffer; followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3).


SFB - BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA - ubiquitin ( Lys48 or Lys63 only ) for 24 h , and MG132 ( 10 μM ) was added for an additional 4 h , lysed with RIPA , subjected to IP using S tag beads followed by Western blot ( n = 3 ) .

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

SFB-BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA-ubiquitin (Lys48 or Lys63 only) for 24 h, and MG132 (10 μM) was added for an additional 4 h, lysed with RIPA, subjected to IP using S tag beads followed by Western blot (n=3).


HeLa cells were co - transfected either wild - type or BRD7 mutant with Flag - PARP1 for 24 h and analysed by Western blot ( n = 3 ) .

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

HeLa cells were co-transfected either wild-type or BRD7 mutant with Flag-PARP1 for 24 h and analysed by Western blot (n=3).


HeLa cells were co - transfected with either wild - type or BRD7 mutant plus HA - ubiquitin for 24 h , and MG132 ( 10 μM ) was added for an additional 4 h and lysed with RIPA buffer , followed by anti - Myc agarose IP and analysed by Western blot with the indicated antibodies ( n = 3 ) .

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

HeLa cells were co-transfected with either wild-type or BRD7 mutant plus HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h and lysed with RIPA buffer, followed by anti-Myc agarose IP and analysed by Western blot with the indicated antibodies (n=3).


HeLa and MDA - MB - 231 cells were transfected with either scrambled or RNF146 siRNAs ( 48 h ) . Protein levels were detected by Western blot . PTEN , which is a known RNF146 - interacting protein , was included as a positive control ( n = 3 ) .

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

HeLa and MDA-MB-231 cells were transfected with either scrambled or RNF146 siRNAs (48 h). Protein levels were detected by Western blot. PTEN, which is a known RNF146-interacting protein, was included as a positive control (n=3).


HeLa cells were transfected with either scrambled or RNF146 siRNAs for 48 h , followed by incubation with 10 μg / ml cycloheximide ( CHX ) for the indicated periods of time . Lysates were harvested and analysed by Western blot ( n = 3 ) . Quantification of BRD7 protein levels from B , n = 3 . Error bars indicate SEM . Relative amounts normalized to the BRD7 protein level at 0 h .

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

HeLa cells were transfected with either scrambled or RNF146 siRNAs for 48 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for the indicated periods of time. Lysates were harvested and analysed by Western blot (n=3). Quantification of BRD7 protein levels from B, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.


Control and RNF146 stably overexpressing HeLa cells were transfected with Myc - BRD7 - WT for 24 h , followed by incubation with 10 μg / ml cycloheximide ( CHX ) for indicated periods of time . Lysates were subjected to Western blot analysis ( n = 3 ) . Quantification of BRD7 protein levels from D , n = 3 . Error bars indicate SEM . Relative amounts normalized to the BRD7 protein level at 0 h .

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

Control and RNF146 stably overexpressing HeLa cells were transfected with Myc-BRD7-WT for 24 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for indicated periods of time. Lysates were subjected to Western blot analysis (n=3). Quantification of BRD7 protein levels from D, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.


HeLa cells transfected with either RNF146 or control siRNA for 24 h followed by transfection of HA - ubiquitin for another 24 h ; MG132 ( 10 μM ) was added for 4 h and lysed with RIPA , followed by anti - BRD7 IP and analysed by Western blot with the indicated antibodies ( n = 3 ) .

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

HeLa cells transfected with either RNF146 or control siRNA for 24 h followed by transfection of HA-ubiquitin for another 24 h; MG132 (10 μM) was added for 4 h and lysed with RIPA, followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3).


HeLa cells stably expressing RNF146 were transfected with HA - ubiquitin for 24 h ; MG132 ( 10 μM ) was added for an additional 4 h , followed by anti - BRD7 IP and Western blot ( n = 3 ) .

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

HeLa cells stably expressing RNF146 were transfected with HA-ubiquitin for 24 h; MG132 (10 μM) was added for an additional 4 h, followed by anti-BRD7 IP and Western blot (n=3).


HeLa cells transfected with the indicated plasmids ; after 24 h , cells were lysed with RIPA buffer followed by immunoprecipitation ( IP ) using either anti - Myc or anti - Flag agarose and Western blot with indicated antibody ( n = 3 ) .

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

HeLa cells transfected with the indicated plasmids; after 24 h, cells were lysed with RIPA buffer followed by immunoprecipitation (IP) using either anti-Myc or anti-Flag agarose and Western blot with indicated antibody (n=3).


HeLa and MDA - MB - 231 cells were lysed with RIPA buffer , and lysates were subjected to IP using either anti - IgG , anti - BRD7 or anti - RNF146 antibodies followed by analysis with Western blot ( n = 3 ) .

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

HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to IP using either anti-IgG, anti-BRD7 or anti-RNF146 antibodies followed by analysis with Western blot (n=3).


Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co - IP using anti - RNF146 antibody . Cell lysates were incubated with protein - G agarose beads conjugated with indicated antibodies , and Western blot was performed ( n = 3 ) .

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

Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co-IP using anti-RNF146 antibody. Cell lysates were incubated with protein-G agarose beads conjugated with indicated antibodies, and Western blot was performed (n=3).


HeLa cells with wild - type or PARP1 knockout were transfected with Myc - BRD7 - WT for 24 h , their lysates were subjected to IP using anti - Myc agarose and analysed by Western blot with indicated antibodies ( n = 3 ) .

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

HeLa cells with wild-type or PARP1 knockout were transfected with Myc-BRD7-WT for 24 h, their lysates were subjected to IP using anti-Myc agarose and analysed by Western blot with indicated antibodies (n=3).


HeLa cells stably expressing Flag - RNF146 were transfected with either Myc - BRD7 - WT or Myc - BRD7 - mutant for 24 h , lysates were subjected to IP using anti - Flag agarose and analysed by Western blot with indicated antibodies ( n = 3 ) .

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

HeLa cells stably expressing Flag-RNF146 were transfected with either Myc-BRD7-WT or Myc-BRD7-mutant for 24 h, lysates were subjected to IP using anti-Flag agarose and analysed by Western blot with indicated antibodies (n=3).


MDA - MB - 231 cells were transfected twice with indicated siRNAs , the absorbance values of indicated cell lines was measured at different time points by CCK8 assay ( n = 3 ) . Data from three independent experiments are expressed as mean ± SEM . Error bars indicate SEM .

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

MDA-MB-231 cells were transfected twice with indicated siRNAs, the absorbance values of indicated cell lines was measured at different time points by CCK8 assay (n=3). Data from three independent experiments are expressed as mean ± SEM. Error bars indicate SEM.


Cell lysates treated in A were analysed by western blot with indicated antibodies ( n = 3 ) .

Cell lysates treated in A were analysed by western blot with indicated antibodies (n=3).