( C D ) amount of p62 located to bodies increases upon inhibition of autophagy . HeLa or S - GFP - p62 HeLa were left untreated or bafilomycin A1 was added for 18 h , were fixed , p62 was either stained red using a p62 mAb ( C ) or imaged directly ( D ) . Nuclei were visualized using Draq5 stain . settings for imaging were identical for treated untreated control .

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(C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S-GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control.


( E ) majority of S - GFP - p62 bodies are stained with antibodies recognizing transiently expressed LC3 . S - GFP - p62 were transiently transfected with myc - LC3 . Myc - LC3 was stained red using an anti - myc tag mAb . boxed indicates part of that is shown to left at a higher magnification . Bars , 5 μm .

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(E) The majority of S-GFP-p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3. S-GFP-p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained red using an anti-myc tag mAb. The boxed area indicates the part of the cell that is shown to the left at a higher magnification. Bars, 5 μm.


( A ) HeLa transiently transfected with GFP - LC3 alone or cotransfected with siRNA against p62 , HA - p62 , or HA - p62 D69A were either left in normal medium or starved for for 1 h . were fixed , p62 was stained using a p62 mAb . More than 200 randomly selected for each condition were scored for pattern of GFP - LC3 as either diffuse or punctuate . frequency of GFP - LC3 - positive with punctuate localization are indicated to right .

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(A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3-positive cells with punctuate localization are indicated to the right.


( B ) Endogenous p62 as well as coexpressed myc - tagged wild - type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP - LC3 from HeLa extracts . GFP or GFP - LC3 was immunoprecipitated from total extracts after cotransfecting indicated constructs . cultures were either left untreated or starved for for 1 h as indicated . Copurified endogenous or ectopically expressed myc - tagged p62 constructs were detected using p62 mAb . Bars , 20 μm .

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(B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.


( A ) HeLa transiently expressing a Flag - tagged NH2 - terminal fragment ( 1 - 171 ) of huntingtin containing a 68 - polyglutamine expansion , Flag - N - HttQ68 . After 48 h , were fixed , Flag - N - HttQ68 was stained green using a Flag mAb . Endogenous p62 was stained red using p62 pAb .

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(A) HeLa cells transiently expressing a Flag-tagged NH2-terminal fragment (amino acids 1-171) of huntingtin containing a 68-polyglutamine expansion, Flag-N-HttQ68. After 48 h, the cells were fixed, and Flag-N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was stained red using the p62 pAb.


( B ) Endogenous p62 forms a shell around aggregated GFP - N - HttQ68 . Transiently transfected HeLa were fixed 48 h after transfection , endogenous p62 was detected using a p62 mAb ( red ) .

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(B) Endogenous p62 forms a shell around aggregated GFP-N-HttQ68. Transiently transfected HeLa cells were fixed 48 h after transfection, and endogenous p62 was detected using a p62 mAb (red).


( C D ) p62 shell surrounding GFP - N - HttQ68 was detected independently of use of antibodies . ( C ) GFP - N - HttQ68 DsRed - tagged p62 were cotransfected into HeLa , of live were obtained after 48 h of expression . left panel shows one confocal plane of a representative GFP - N - HttQ68 DsRed - p62 double - positive structure , right panel shows Z - stack of planes with side views of aggregate at side at top . N , nucleus . ( A - C ) Nuclei were detected using Draq5 . ( D ) GFP - N - HttQ68 tdTomato - tagged p62 were cotransfected into HeLa , of live were obtained after 24 h of expression .

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(C and D) The p62 shell surrounding GFP-N-HttQ68 was detected independently of the use of antibodies. (C) GFP-N-HttQ68 and DsRed-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 48 h of expression. The left panel shows one confocal plane of a representative GFP-N-HttQ68 and DsRed-p62 double-positive structure, and the right panel shows the Z-stack of planes with side views of the aggregate at the side and at the top. N, nucleus. (A-C) Nuclei were detected using Draq5. (D) GFP-N-HttQ68 and tdTomato-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 24 h of expression.


( E ) Transiently expressed Flag - N - HttQ68 myc - LC3 colocalize in GFP - p62 - positive structures . S - GFP - p62 were fixed 48 h after transfection , Flag - N - HttQ68 was stained red using a Flag tag mAb , myc - LC3 was stained far red ( blue ) using a chicken myc tag pAb ( top ) . bottom panel shows a live image of a GFP - N - HttQ68 aggregate surrounded by a shell containing tdTomato - LC3 . Bars , 5 μm .

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(E) Transiently expressed Flag-N-HttQ68 and myc-LC3 colocalize in GFP-p62-positive structures. S-GFP-p62 cells were fixed 48 h after transfection, Flag-N-HttQ68 was stained red using a Flag tag mAb, and myc-LC3 was stained far red (blue) using a chicken myc tag pAb (top). The bottom panel shows a live cell image of a GFP-N-HttQ68 aggregate surrounded by a shell containing tdTomato-LC3. Bars, 5 μm.


( A ) GFP - positive were scored as live or apoptotic by appearance of Draq5 . After 48 h of expression , GFP - positive attached to surface with a normal appearance ( open arrowheads ) were scored as live , whereas rounded , partially detached with a condensed or fragmented nuclei ( arrows ) were scored as dead . More than 200 GFP - positive in randomly selected microscope views were scored . Bars , 20 μm .

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(A) GFP-positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h of expression, GFP-positive cells attached to the surface with a normal nuclear appearance (open arrowheads) and were scored as live, whereas rounded, partially detached cells with a condensed or fragmented nuclei (arrows) were scored as dead. More than 200 GFP-positive cells in randomly selected microscope views were scored. Bars, 20 μm.


( B ) effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking UBA domain on N - HttQ68 - induced in HeLa SHSY - 5Y neuroblastoma .

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(B) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N-HttQ68-induced cell death in HeLa and SHSY-5Y neuroblastoma cells.


G ) Representative of EdU in freshly isolated YC OC FAPs following EdU in vivo exposure for 3 days ( EdU in red / DAPI in blue ) . Scale bar = 25 μm . H ) Graph showing of EdU positive for shown in ( G ) .

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G) Representative images of the EdU staining in freshly isolated YC and OC FAPs following EdU in vivo exposure for 3 days (EdU in red/DAPI in blue). Scale bar = 25 μm. H) Graph showing the percentage of EdU positive cells for the staining shown in (G).


I ) Representative of LMNB1 in freshly isolated YC OC FAPs ( LMNB1 in green / DAPI in blue ) . Scale bar = 25 μm . J ) Graph showing relative of LMNB1 normalized for nuclei number , in shown in ( I ) .

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I) Representative images of the LMNB1 staining in freshly isolated YC and OC FAPs (LMNB1 in green/DAPI in blue). Scale bar = 25 μm. J) Graph showing the relative quantification of LMNB1 fluorescence intensity normalized for nuclei number,in the staining shown in (I).


L ) Representative of γH2AX KI - 67 in freshly isolated YC OC FAPs ( γH2AX in green / KI - 67 in red / DAPI in blue ) . Scale bar = 10 μm . Magnification in insert ( scale bar = 3 μm ) shows γH2AX positive CCF in OC FAPs . M ) Graph showing of positive for γH2AX positive CCF , for shown in ( L ) . N ) Graph showing of positive for KI - 67 for shown in ( L ) .

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L) Representative images of the γH2AX and KI-67 staining in freshly isolated YC and OC FAPs (γH2AX in green/KI-67 in red/DAPI in blue). Scale bar = 10 μm. Magnification in the insert (scale bar = 3 μm) shows γH2AX positive CCF in OC FAPs. M) Graph showing the percentage of cells positive for γH2AX positive CCF, for the staining shown in (L). N) Graph showing the percentage of cells positive for KI-67 for the staining shown in (L).


D ) Representative of phospho - NFkB in FAPs ( pNFkB - p65 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal section of YC , YT , OC OT mice ; Scale bar = 25 μm ; E ) Box plot showing of pNFkB - p65 positive FAPs in shown in ( D )

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D) Representative images of the phospho-NFkB staining in FAPs (pNFkB-p65 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal section of YC, YT, OC and OT mice; Scale bar = 25 μm; E) Box plot showing the percentage of pNFkB-p65 positive FAPs in the staining shown in (D)


F ) Representative of phospho - p38 in FAPs ( pP38 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal sections of YC , YT , OC OT mice . Scale bar = 25 μm ; G ) Box plot showing of pP38 positive FAPs in shown

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F) Representative images of the phospho-p38 staining in FAPs (pP38 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; G) Box plot showing the percentage of pP38 positive FAPs in the staining shown in


H ) Representative of phospho - SMAD2 / 3 in FAPs ( pSMAD2 / 3 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal sections of YC , YT , OC OT mice . Scale bar = 25 μm ; I ) Box plot showing of pSMAD2 / 3 positive FAPs in shown in ( H ) .

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H) Representative images of the phospho-SMAD2/3 staining in FAPs (pSMAD2/3 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; I) Box plot showing the percentage of pSMAD2/3 positive FAPs in the staining shown in (H).


Pulldowns of pointed end site 1 - 4 mutants using Strep - Hook3 ( D ) or Strep - BICD2 ( E ) . Binding is shown relative to binding of wildtype pointed end construct ( dashed lines ) . * shows p < 0 . 05 . For BICD2 , p = 0 . 06 for Site 1 mutant , p = 0 . 1 for Site 4 mutant . Data presented as mean ± , n = 3 . Statistical significance calculated using ANOVA with Tukey ' s for multiple comparisons .

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Pulldowns of pointed end site 1-4 mutants using Strep-Hook3 (D) or Strep-BICD2 (E). Binding is shown relative to the binding of the wildtype pointed end construct (dashed lines). * shows p<0.05. For BICD2, p=0.06 for Site 1 mutant, and p=0.1 for Site 4 mutant. Data presented as mean ±SEM, n=3. Statistical significance calculated using ANOVA with Tukey's test for multiple comparisons.


( A ) Representative ( details from boxed regions ) of WT MEF transfected with YFPParkin ( green ) treated with FCCP ( 10 μM ) to induce mitophagy . were immunostained with anti - Tom20 ( magenta ) to reveal mitochondrial network . Each row represents one stage in process of mitophagy that has been used to score progression of mitophagy at different time - points for different conditions tested ( specific time - point of each example is indicated in image ) . From top to bottom : i ) " Diffuse Parkin " defines in where Parkin expression is homogenously distributed throughout cytoplasm ( quantified in B ) . ii ) Soon after mitochondrial damage Parkin appears enriched in small " " throughout cytoplasm localizing within isolated mitochondria ( quantified in C ) . iii ) Later in process these structures aggregate in bigger structures accumulating in perinuclear regions ( quantified in D ) . iv ) process of Parkin translocation eventually affects mitochondrial population usually collapses in few large structures possibly forming part of big autolysosomes in perinuclear region ( quantified in D ) . Scoring was performed from 3 independent experiments ( n = 3 ; one way ANOVA with Dunnett post ) . Error bars represent s . e . m . Significance : * p < 0 . 05 * * p < 0 .

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(A) Representative images (and details from boxed regions) of WT MEF cells transfected with YFPParkin (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one stage in the process of mitophagy that has been used to score the progression of mitophagy at different time-points for the different conditions tested (the specific time-point of each example is indicated in the image). From top to bottom: i) "Diffuse Parkin" defines cells in where Parkin expression is homogenously distributed throughout the cytoplasm (quantified in B). ii) Soon after mitochondrial damage Parkin appears enriched in small "puncta" throughout the cytoplasm localizing within isolated mitochondria (quantified in C). iii) Later in the process these puncta structures aggregate in bigger structures accumulating in perinuclear regions (quantified in D). iv) The process of Parkin translocation eventually affects all the mitochondrial population and usually collapses in few large structures possibly forming part of big autolysosomes in the perinuclear region (quantified in D). Scoring was performed from 3 independent experiments (n=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01


( F ) Representative ( details from boxed regions ) of WT MEFs transfected with GFPPINK1 ( green ) treated with FCCP ( 10 μM ) to induce mitophagy . were immunostained with anti - Tom20 ( magenta ) to reveal mitochondrial network . Each row represents one of three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage ( , intermediate mitochondrial ) . scoring of these categories was used to describe PINK1 translocation to mitochondria in WT MiroDKO at different time - points shortly after mitochondrial insult ( G ) . Data collected from 3 independent experiments ( n = 3 ) . Error bars represent s . e . m . Significance : * p < 0 . 05 * * p < 0 . 01

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(F) Representative images (and details from boxed regions) of WT MEFs cells transfected with GFPPINK1 (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one of the three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and MiroDKO cells at different time-points shortly after mitochondrial insult (G). Data collected from 3 independent experiments (n=3). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01


B ) Representative showing full control selected Miro1 lysine mutants ( Miro15R , Miro1allR Miro1R572K ) expressing in MiroDKO MEF . constructs ( myc - tag , green ) localise to mitochondria ( Tom20 , red ) .

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B) Representative images showing full length control and selected Miro1 lysine mutants (Miro15R, Miro1allR and Miro1R572K) expressing in MiroDKO MEF cells. All constructs (myc-tag, green) localise to mitochondria (Tom20, red).


C ) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH - SY5Y .

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C) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH-SY5Y cells.


D ) Ubiquitination showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment ( 1h , 10 μM ) in FlagParkin overexpressing SH - SY5Y . effect is quantified in ( E ) ( n = 4 independent experiments for Miro1WT Miro15R n = 3 for Miro1allR Miro1R572K , ANOVA with Sidak ' s post hoc ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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D) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment (1h, 10 μM) in FlagParkin overexpressing SH-SY5Y. The effect is quantified in (E) (n=4 independent experiments for Miro1WT and Miro15R and n=3 for Miro1allR and Miro1R572K, ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


F ) Representative western blot showing a degradation in FlagParkin overexpressing SH - SY5Y transfected with Miro1WT , Miro15R or Miro1allR constructs treated with FCCP ( 10 μM ) for 3 or 6 hours . of Miro1 ( G ) matrix PDH E1α in ( H ) Parkin ( I ) ( n = 4 independent experiments ; ANOVA with Sidak ' s post hoc ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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F) Representative western blot showing a degradation assay in FlagParkin overexpressing SH-SY5Y cells transfected with Miro1WT, Miro15R or Miro1allR constructs and treated with FCCP (10 μM) for 3 or 6 hours. Quantification of Miro1 levels (G) the matrix protein PDH E1α in (H) and Parkin levels (I) (n=4 independent experiments; ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative at 3 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO co - transfected with specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal mitochondrial network . A detail of YFPParkin translocation onto mitochondrial network is also shown for each condition .

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(A) Representative images at 3 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. A detail of YFPParkin translocation onto the mitochondrial network is also shown for each condition.


( B C ) of fraction of showing advanced stages of mitophagic process ( showing mitochondrial aggregation due to advanced Parkin translocation or complete translocation of Parkin onto mitochondrial network ) at 1 hour ( B ) or 3 hours ( C ) after FCCP treatment ( data collected from at least 3 independent experiments : WT n = 7 , MiroDKO n = 8 , + mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; mycMiro2 = 3 ; one way ANOVA with Dunnett post ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(B and C) Quantification of the fraction of cells showing advanced stages of the mitophagic process (cells showing mitochondrial aggregation due to advanced Parkin translocation or the complete translocation of Parkin onto all the mitochondrial network) at 1 hour (B) or 3 hours (C) after FCCP treatment (data collected from at least 3 independent experiments: WT n=7, MiroDKO n=8, +mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; mycMiro2=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


D ) Western blots of Miro1 degradation in MiroDKO co - expressing YFPParkin indicated myc - tagged Miro1 constructs treated with FCCP ( 10 μM ) for indicated times ( n = 3 different experiments ; 2 - way - ANOVA with Dunnett post ; comparisons between Miro1WT Miro1allR are represented with yellow asteriscs , comparisons between Miro1WT Miro15R are represented in green comparisons between Miro15R Miro1allR are represented in black ) .

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D) Western blots and quantification of Miro1 degradation in MiroDKO cells co-expressing YFPParkin and the indicated myc-tagged Miro1 constructs and treated with FCCP (10 μM) for the indicated times (n=3 different experiments; 2-way-ANOVA with Dunnett post test; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs, comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black).


E ) . Representative at 24 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO co - transfected with specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal mitochondrial network . Expression of both ubiquitin mutants ( Miro15R Miro1allR - arrowheads ) protects from mitophagic clearance of mitochondria ( arrows ) after damage . ( F ) of fraction of showing complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment ( data collected from at least 3 independent experiments : MiroDKO n = 8 , mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; one way ANOVA with Dunnett post ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001

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E). Representative images at 24 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. Expression of both ubiquitin mutants (Miro15R and Miro1allR - arrowheads) protects from the mitophagic clearance of mitochondria (arrows) after damage. (F) Quantification of the fraction of cells showing the complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment (data collected from at least 3 independent experiments: MiroDKO n=8, mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative confocal of soma of WT Miro1KO cortical neurons expressing YFPParkin MtDsRed after valinomycin treatment at indicated time points ( scale bars = 5 µm ) .

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(A) Representative confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed after valinomycin treatment at the indicated time points (scale bars = 5 µm).


( B ) Airyscan confocal of Parkin recruitment after 5 hours of valinomycin treatment in WT Miro1KO neuronal processes ( scale bars = 2 . 5 µm ) .

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(B) Airyscan confocal images of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal processes (scale bars = 2.5 µm).


( C ) linescans of YFPParkin MtDsRed signal in WT Miro1KO neurons after 5 hours of valinomycin treatment ( lines shown in A ) .

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(C) Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment (lines shown in A).


( D ) of Parkin recruitment to mitochondria ( YFPParkin signal overlapping MtDsRed signal - - adjusted normalised to t = 0 ) in WT Miro1KO neurons following valinomycin treatment ( n = 15 conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(D) Quantification of Parkin recruitment to mitochondria (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted and normalised to t=0) in WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( E ) of mitochondrial occupancy ( of MtDsRed signal in soma / entire of soma ) in somas of WT Miro1KO neurons following valinomycin treatment ( n = 15 conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) .

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(E) Quantification of mitochondrial occupancy (area of MtDsRed signal in soma / entire area of soma) in the somas of WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA).


( F ) of mitochondrial clearance in somas of WT Miro1KO neurons between 2 5 hours of valinomycin treatment ( n = 15 conditions per genotype over 3 neuronal preparations ; Unpaired t - ) .

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(F) Quantification of mitochondrial clearance in somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; Unpaired t-test).


( G ) Representative confocal of WT , Miro1KO PINK1KO cortical neurons immunostained with MAP2 ( cyan ) s65 - phospho - ubiquitin ( green ) after valinomycin treatment ( Scale bars = 20µm ) . ( H ) of s65 - phospho - ubiquitin signal within MAP2 signal ( normalised to MAP2 t = 0 ) in Miro1WT , Miro1KO PINK1KO neurons following valinomycin treatment n = 3 , 4 3 from WT , Miro1KO PINK1KO , respectively ( 6 ROIs per condition ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(G) Representative confocal images of WT, Miro1KO and PINK1KO cortical neurons immunostained with MAP2 (cyan) and s65-phospho-ubiquitin (green) after valinomycin treatment (Scale bars = 20µm). (H) Quantification of s65-phospho-ubiquitin signal intensity within MAP2 signal (normalised to MAP2 area and t=0) in Miro1WT, Miro1KO and PINK1KO neurons following valinomycin treatment n= 3, 4 and 3 embryos from WT, Miro1KO and PINK1KO embryos, respectively (6 ROIs per condition; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( I ) Representative confocal of soma of Miro1KO cortical neurons expressing Miro1WT mutant forms of Miro1 , YFPParkin MtDsRed without valinomycin treatment ( Scale bars = 10 µm ) . of parkin colocalization with MtDsRed ( YFPParkin signal overlapping MtDsRed signal - - adjusted ) in soma of Miro1KO PINK1KO cortical neurons ( n = 12 conditions per genotype over 3 neuronal preparations ; one - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(I) Representative confocal images of the soma of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1, YFPParkin and MtDsRed without valinomycin treatment (Scale bars = 10 µm). Quantification of parkin colocalization with MtDsRed (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted) in the soma of Miro1KO and PINK1KO cortical neurons (n=12 cells all conditions per genotype over 3 neuronal preparations; one-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( J ) Representative confocal of soma of Miro1KO cortical neurons expressing WT mutant forms of Miro1 , YFPParkin MtDsRed after 5 hours of valinomycin treatment ( Scale bars = 10 µm ) . of Parkin recruitment to mitochondria ( Normalised to t = 0 ) in Miro1KO PINK1KO cortical neurons following valinomycin treatment ( n = 12 conditions per genotype over 3 neuronal preparations , two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001

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(J) Representative confocal images of the soma of Miro1KO cortical neurons expressing WT and mutant forms of Miro1, YFPParkin and MtDsRed after 5 hours of valinomycin treatment (Scale bars = 10 µm). Quantification of Parkin recruitment to mitochondria (Normalised to t=0) in Miro1KO and PINK1KO cortical neurons following valinomycin treatment (n=12 cells all conditions per genotype over 3 neuronal preparations, two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative blots of 4 12 months old hippocampal lysates from control , Miro1CKO Miro2KO . B ) of in 4 month lysates 12 month lysates ( n = 3 / genotype at 4 months n = 4 / genotype at 12 months ; one - way ANOVA with post - hoc Dunnett ' s ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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(A) Representative blots of 4 and 12 months old hippocampal lysates from control, Miro1CKO and Miro2KO. B) Quantification of protein levels in 4 month lysates and 12 month lysates (n=3 animals / genotype at 4 months and n=4 animals / genotype at 12 months; one-way ANOVA with post-hoc Dunnett's test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


C ) Representative of hippocampal regions from control Miro1CKO crossed with MitoDendra .

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C) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals.


( D ) Details from cortical regions of control , Miro1CKO Miro2KO at 12 months of showing large mitochondrial structures occurring only in case of Miro1CKO .

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(D) Details from cortical regions of control, Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals.


( E ) Electron microcopy of control Miro1CKO brains showing ultrastructure of mitochondrial compartment . mitochondrial units in Miro1CKO are enlarged show altered cristae structure at 4 months of . This process is progressive as mitochondrial structures appear larger with reduced intramitochondrial complexity at 12 months of .

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(E) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment. The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age. This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age.


Representative ( F ) ( G ) of Mfn1 Mfn2 in cortical slices from 12 month WT Miro1CKO mice ( n = 4 / genotype ; student ' s t - ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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Representative images (F) and quantification (G) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice (n=4 animals / genotype; student's t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative western blot of brain lysates from 12 - month WT , Miro1CKO Miro2KO mice immunoblotted with antibodies stated . ( B ) of eIF2α P - eIF2α band ( normalised to actin band ) from 12 - month WT , Miro1CKO Miro2KO brain lysates . Phosphorylation of eIF2α is defined as P - eIF2α / eIF2α ( n = 4 mice per genotype , unpaired t - ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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(A) Representative western blot images of brain lysates from 12-month WT, Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated. (B) Quantification of eIF2α and P-eIF2α band intensity (normalised to actin band intensity) from 12-month WT, Miro1CKO and Miro2KO brain lysates. Phosphorylation of eIF2α is defined as P-eIF2α/eIF2α (n=4 mice per genotype, unpaired t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( C ) Representative confocal of cortical regions of 12 - month mitoDendra - crossed WT Miro1CKO mice stained with MAP2 P - eIF2α . ( D ) of P - eIF2α signal from cortical regions of 12 - month mitoDendra - crossed WT Miro1CKO mice ( n = 4 mice per genotype ; unpaired t - ) .

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(C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice (n=4 mice per genotype; unpaired t-test).


( E ) Representative confocal ( 63x ) of cortical regions of 12 - month mitoDendra - crossed WT Miro1CKO mice stained with MAP2 P - eIF2α . Arrows indicate presence of megamitochondria ( > 2 . 5 µm2 ) , stars indicate neuronal somas without megamitochondria .

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(E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without megamitochondria.


( F ) Example zoom of neuronal somas with without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO mice stained with P - eIF2α . ( G ) P - eIF2α signal ( a . u . ) from neuronal somas with without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO ( n = 16 sections from 4 mice ; paired t - ) .

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(F) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).


A Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 54 ) close contacts ( n = 86 ) were measured by surrogate neutralization ( sVNT ) . inhibition of ACE2 binding to RBD at plasma dilution of 1 : 800 is given . wild type ( Wuhan ) or Delta ( B . 1 . 617 . 2 ) RBD was used respectively .

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A Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=54) and close contacts (n=86) were measured by surrogate virus neutralization test (sVNT). The percentage inhibition of ACE2 binding to RBD at plasma dilution of 1:800 is given. The wild type (Wuhan) strain or Delta (B.1.617.2) strain RBD was used respectively.


B Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 29 ) close contacts ( n = 86 ) were measured by pseudovirus neutralization .

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B Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=29) and close contacts (n=86) were measured by pseudovirus neutralization assay.


C Anti - SARS - CoV - 2 S antibodies in plasma of vaccine breakthrough cases ( n = 48 ) close contacts ( n = 86 ) was determined by S flow cytometry - ( SFB ) . of plasma antibody - bound out of total population of transfected with - anchored SARS - CoV - 2 S is given . In graphs error bars denote median interquartile range . Red solid circles indicate Delta vaccine breakthrough , red open circles indicate non - Delta or unknown .

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C Anti-SARS-CoV-2 S protein antibodies in plasma of vaccine breakthrough cases (n=48) and close contacts (n=86) was determined by S protein flow cytometry-based assay (SFB). The percentage of plasma antibody-bound cells out of the total population of cells transfected with membrane-anchored SARS-CoV-2 S protein is given. In all graphs error bars denote median and interquartile range. Red solid circles indicate Delta strain vaccine breakthrough, red open circles indicate non-Delta or unknown strain.


B , C of anti - SARS - CoV - 2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate neutralization ( sVNT ) , in supernatant from 5 - day culture of activated memory B in plasma . difference in sVNT % inhibition between WT Delta is taken , this difference is compared between plasma memory B supernatant for vaccine breakthrough cases ( n = 48 pairs ) ( B ) , for close contacts ( n = 86 pairs ) ( C ) .

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B, C Levels of anti-SARS-CoV-2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate virus neutralization test (sVNT), in supernatant from 5-day culture of activated memory B cells and in plasma. The difference in sVNT % inhibition between WT and Delta is taken, and this difference is compared between plasma and memory B cell supernatant for vaccine breakthrough cases (n=48 pairs) (B), and for close contacts (n=86 pairs) (C).


A - D PBMCs from vaccine breakthrough cases ( n = 15 ) close contacts ( n = 26 ) were examined for T responses . PBMCs were left unstimulated ( Unstim ) , were stimulated with pooled SARS - CoV - 2 PepTivator ® S , S1 , M N peptides for 6 hours ( SARS - CoV - 2 Peptide Stim ) , or non - specifically stimulated with phorbol 12 - myristate 13 - acetate ( PMA ) , then assessed by high - dimensional flow cytometry . CD4 + T positive for intracellular of IFNγ ( a ) , IL - 2 ( b ) , TNF ( c ) , or CD8 + T positive for intracellular of granzyme B ( d ) are shown . Error bars indicate median interquartile range .

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A-D PBMCs from vaccine breakthrough cases (n=15) and close contacts (n=26) were examined for T cell responses. PBMCs were left unstimulated (Unstim), were stimulated with pooled SARS-CoV-2 PepTivator® S, S1, M and N peptides for 6 hours (SARS-CoV-2 Peptide Stim), or non-specifically stimulated with phorbol 12-myristate 13-acetate (PMA), then assessed by high-dimensional flow cytometry. CD4+ T cells positive for intracellular staining of IFNγ (a), IL-2 (b), TNF (c), or CD8+ T cells positive for intracellular staining of granzyme B (d) are shown. Error bars indicate median and interquartile range.


F differentiation status of SARS - CoV - 2 peptide - stimulated CD4 + T ( left ) CD8 + T ( right ) were compared on CD27 CD45RA expression ( Naïve : CD27 + CD45RA + ; T central memory ( TCM ) : CD27 + CD45RA - ; T effector memory ( ) : CD27 - CD45RA - ; TEMRA : CD27 - CD45RA + ) . average of vaccine breakthrough cases or close contacts are plotted respectively .

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F The differentiation status of SARS-CoV-2 peptide-stimulated CD4+ T cells (left) and CD8+ T cells (right) were compared based on CD27 and CD45RA expression (Naïve: CD27+ CD45RA+; T central memory (TCM): CD27+ CD45RA-; T effector memory (TEM): CD27- CD45RA-; TEMRA: CD27- CD45RA+). The average of all vaccine breakthrough cases or close contacts are plotted respectively.


B of selected immune mediator in plasma samples of healthy controls , vaccinated close contacts , COVID - 19 vaccine breakthrough cases , unvaccinated COVID - 19 with infection . Each color represents relative of a particular analyte . Blue red indicate low high , respectively .

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B Heatmap of selected immune mediator levels in plasma samples of healthy controls, vaccinated close contacts, COVID-19 vaccine breakthrough cases, and unvaccinated COVID-19 patients with primary infection. Each color represents the relative concentration of a particular analyte. Blue and red indicate low and high concentration, respectively.


( B ) upper panel showing qRT - PCR of four identified LUAD - associated RBPs ( ADARB1 , CELF2 , QKI ZFP36 ) mRNA in 61 LUAD tissues matched paracarcinoma tissues . lower panel showing relative mRNA expression ( T / N ) of four RBPs in metastatic ( n = 28 ) non - metastatic ( n = 33 ) LUAD tissues . LUAD tissues were classified into metastatic non - metastatic tissues as described in Materials Methods . T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents log10 transformed fold change of mRNA expression ( T / N ) of four RBPs .

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(B) The upper panel showing qRT-PCR analysis of four identified LUAD-associated RBPs (ADARB1, CELF2, QKI and ZFP36) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues. The lower panel showing relative mRNA expression (T/N) of the four RBPs in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. LUAD tissues were classified into metastatic and non-metastatic tissues as described in Materials and Methods. T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression (T/N) of four RBPs.


( E - G ) Kaplan - Meier survival curves of LC , LUAD LUSC ( n = 1540 , n = 865 n = 675 , respectively ) with high or low expression of QKI . log - rank was used to analyze difference between two groups .

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(E-G) Kaplan-Meier survival curves of LC, LUAD and LUSC patients (n=1540, n=865 and n=675, respectively) with high or low expression levels of QKI. The log-rank test was used to analyze the difference between two groups.


( H ) qRT - PCR of endogenous mRNA of QKI - 5 , QKI - 6 , QKI - 7 in human bronchial epithelial BEAS - 2B , LUAD lines A549 H1299 .

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(H) qRT-PCR analysis of endogenous mRNA levels of QKI-5, QKI-6, and QKI-7 in human bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299.


( J ) In vitro pull - down assays were performed using biotin - labeled TGFβR1 3 ' UTR segments in A549 H1299 . After pull - down , endogenous QKI - 5 enrichments were detected by western blot analyses . β - actin was used as control .

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(J) In vitro RNA pull-down assays were performed using biotin-labeled TGFβR1 3' UTR segments in A549 and H1299 cells. After pull-down, endogenous QKI-5 enrichments were detected by western blot analyses. β-actin was used as the protein control.


( A B ) qRT - PCR western blot analyses of TGFβR1 mRNA in QKI - 5 - overexpressing A549 H1299 . ( C D ) Expression of TGFβR1 mRNA in QKI - 5 - silenced A549 H1299 .

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(A and B) qRT-PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI-5-overexpressing A549 and H1299 cells. (C and D) Expression of TGFβR1 mRNA and protein in QKI-5-silenced A549 and H1299 cells.


( K ) QKI - 5 - overexpressing A549 H1299 were treated subjected to qRT - PCR assays to detect mRNA expression of downstream genes of TGF - β / SMAD signaling , including PAI - 1 , Slug , Snail , E - cadherin , / or N - cadherin .

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(K) QKI-5-overexpressing A549 and H1299 cells were treated and subjected to qRT-PCR assays to detect the mRNA expression of downstream genes of the TGF-β/SMAD signaling, including PAI-1, Slug, Snail, E-cadherin, and/or N-cadherin.


( C - F ) Wound - healing migration assays were performed on QKI - 5 - overexpressing A549 H1299 as well as control wound healing was recorded ( C E ) quantitatively measured ( D F ) at least six times .

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(C-F) Wound-healing migration assays were performed on QKI-5-overexpressing A549 and H1299 cells as well as control cells The wound healing was recorded (C and E) and quantitatively measured (D and F) at least six times.


( G H ) In presence or absence of TGF - β1 , QKI - 5 - overexpressing A549 H1299 were allowed to migrate through a polycarbonate in Transwells . After 24 h , migrated were stained , photographed counted in at least four random fields under a microscope . Representative ( left ) migrated numbers ( right ) were presented . ( I J ) QKI - 5 - overexpressing A549 H1299 were treated allowed to invade through Matrigel - coated in Transwells . Invasive were

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(G and H) In the presence or absence of TGF-β1, QKI-5-overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells. After 24 h, migrated cells were stained, photographed and counted in at least four random fields under a light microscope. Representative images (left) and the migrated cell numbers (right) were presented. (I and J) QKI-5-overexpressing A549 and H1299 cells were treated and allowed to invade through Matrigel-coated membrane in Transwells. Invasive cells were determined


( E ) H & E was performed for evaluation of lung micrometastases . Representative showing micrometastases of lung tissues from a pair of mice Blue red arrowheads indicate lung micrometastases of vector group QKI - 5 - overexpressing group , respectively . ( F ) Dot plots showing difference of micrometastases in lung tissues between QKI - 5 - overexpressing group vector control group ( n = 10 mice per group ) . ( G ) Representative microscopic of H & E for liver micrometastases in a pair of mice Blue red arrowheads indicate liver micrometastases . ( H ) Dot plots showing difference of micrometastases in liver tissues between QKI - 5 - overexpressing group control vector group ( n = 10 mice per group ) .

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(E) H&E staining was performed for the evaluation of lung micrometastases. Representative images showing micrometastases of lung tissues from a pair of mice Blue and red arrowheads indicate lung micrometastases of vector group and QKI-5-overexpressing group, respectively. (F) Dot plots showing the difference of the micrometastases counts in lung tissues between QKI-5-overexpressing group and vector control group (n = 10 mice per group). (G) Representative microscopic photographs of H&E staining for liver micrometastases in a pair of mice Blue and red arrowheads indicate liver micrometastases. (H) Dot plots showing the difference of the micrometastases counts in liver tissues between QKI-5-overexpressing group and control vector group (n = 10 mice per group).


( C - F ) In presence or absence of TGF - β1 , migratory invasive capabilities of above - treated A549 H1299 were evaluated by Transwell assays Migrated invasive were stained counted under a microscope .

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(C-F) In the presence or absence of TGF-β1, the migratory and invasive capabilities of above-treated A549 and H1299 cells were evaluated by Transwell assays Migrated and invasive cells were stained and counted under a light microscope.


( J ) Kaplan - Meier survival curves of LUAD ( n = 865 ) with high or low expression of TGFβR1 . log - rank was used to compare difference between two groups .

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(J) Kaplan-Meier survival curves of LUAD patients (n=865) with high or low expression levels of TGFβR1. The log-rank test was used to compare the difference between two groups.


qRT - PCR analyses of mRNA expression of QKI - 5 in A549 transfected with siRNAs of TFs , which have potential binding sites in QKI - 5 promoter .

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qRT-PCR analyses of mRNA expression of QKI-5 in A549 cells transfected with siRNAs of TFs, which have potential binding sites in QKI-5 promoter.


( E ) Western blot of QKI - 5 in KLF6 - silenced A549 .

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(E) Western blot analysis of QKI-5 protein levels in KLF6-silenced A549 cells.


( F ) An overview of KLF6 - ChIP - seq data ( GSE96355 ) is illustrated using Integrative Genomics Viewer ( IGV ) software . An obvious peak was detected at position - 100 to + 100 in QKI - 5 promoter .

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(F) An overview of KLF6-ChIP-seq data (GSE96355) is illustrated using Integrative Genomics Viewer (IGV) software. An obvious peak was detected at position -100 to +100 in the QKI-5 promoter.


qRT - PCR of KLF6 mRNA in 61 LUAD tissues matched paracarcinoma tissues ( I ) T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents log10 transformed fold change of mRNA expression of KLF6 . Central dotted of violin plot represents median , upper lower dotted represents quartile ( I ) .

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qRT-PCR analysis of KLF6 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (I) T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of KLF6. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile (I).


( A ) Western blot of supernatants containing long short mouse human DNGR - 1 ECD under reducing non - reducing conditions .

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(A) Western blot analysis of supernatants containing long and short mouse and human DNGR-1 ECD proteins under reducing and non-reducing conditions.


( B ) Western blot of supernatants after production of WT C94S mutant long mouse DNGR - 1 ECD under reducing non - reducing conditions . C94S mutant - containing supernatant was 200x concentrated before .

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(B) Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR-1 ECD proteins under reducing and non-reducing conditions. C94S mutant-containing supernatant was 200x concentrated before analysis.


( C ) Western blot of supernatants containing indicated DNGR - 1 ECD after treatment with PNGase F overnight . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers .

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(C) Western blot analysis of supernatants containing the indicated DNGR-1 ECD proteins after treatment with PNGase F overnight. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers.


( A ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into 10 mM Tris buffer of indicated pH ( left panel ) or into 10 mM Tris pH 7 . 4 buffer supplemented with increasing amounts of NaCl ( 1 - 250 mM ; right panel ) , reduction sensitivity of DNGR - 1 was assessed by reducing SDS - PAGE Western blot . ( B ) 0 . 25 μg of purified DNGR - 1 long mouse ECD was diluted into buffers of different pH ionic strength , its reduction sensitivity was assessed by reducing SDS - PAGE Western blot . of bands corresponding to dimer monomer was determined densitometrically , was plotted as a function of buffer ionic strength pH .

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(A) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into 10 mM Tris buffer of the indicated pH (left panel) or into 10 mM Tris pH 7.4 buffer supplemented with increasing amounts of NaCl (1 - 250 mM; right panel), and reduction sensitivity of DNGR-1 was assessed by reducing SDS-PAGE and Western blot. (B) 0.25 μg of purified DNGR-1 long mouse ECD was diluted into buffers of different pH and ionic strength, and its reduction sensitivity was assessed by reducing SDS-PAGE and Western blot. The intensity of bands corresponding to dimer and monomer was determined densitometrically, and the ratio was plotted as a function of buffer ionic strength and pH.


( C ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffer , its reduction sensitivity was tested under mildly ( Laemmli buffer ) or strongly denaturing ( 8 M urea ) conditions by reducing SDS - PAGE Western blot .

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(C) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffer, and its reduction sensitivity was tested under mildly (Laemmli buffer) or strongly denaturing (8 M urea) conditions by reducing SDS-PAGE and Western blot.


( D ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffers , its reduction sensitivity after different lengths of heat - denaturation in Laemmli buffer was tested by reducing SDS - PAGE Western blot . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(D) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffers, and its reduction sensitivity after different lengths of heat-denaturation in Laemmli buffer was tested by reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) 1 μg of purified long mouse DNGR - 1 ECD was transferred into 10mM MES pH 6 . 1 0 . 5 μg into PBS . Half of MES sample whole PBS sample were dialyzed against 2 l of PBS overnight at 4 ° C . After dialysis , samples were prepared for reducing SDS - PAGE Western blot .

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(A) 1 μg of purified long mouse DNGR-1 ECD was transferred into 10mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS-PAGE and Western blot.


( B ) Supernatants after production of indicated in 293F were harvested diluted into indicated buffers at a 2 : 3 analyzed using reducing SDS - PAGE Western blot . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(B) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2:3 ratio and analyzed using reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( B ) Supernatants containing ECD of long mouse DNGR - 1 , Dectin - 1 chimeric receptor were prepared for SDS - PAGE Western blot under non - reducing conditions .

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(B) Supernatants containing ECD of long mouse DNGR-1, Dectin-1 and the chimeric receptor were prepared for SDS-PAGE and Western blot under non-reducing conditions.


( C ) Supernatants containing indicated were diluted into PBS or pH 6 . 1 MES buffer analyzed using SDS - PAGE Western blot under reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction sensitive with a + .

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(C) Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS-PAGE and Western blot under reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction sensitive protein with a +.


( A ) Long mouse human DNGR - 1 ECD were diluted in PBS or 10 mM MES pH 6 . 1 buffer 20 independent far - UV spectra were acquired for each condition ( red black lines depict composite curve for each condition )

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(A) Long mouse and human DNGR-1 ECD proteins were diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent far-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition)


( B ) Long mouse DNGR - 1 ECD was diluted in PBS or 10 mM MES pH 6 . 1 buffer 20 independent near - UV spectra were acquired for each condition ( red black lines depict composite curve for each condition blue shows difference between two ) .

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(B) Long mouse DNGR-1 ECD was diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent near-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition and the blue line shows the difference between the two).


( B ) ( C ) Supernatants containing indicated ECD ( mouse , long isoform ) were diluted 1 : 2 into indicated buffers analyzed by SDS - PAGE Western blot under reducing or non reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(B) and (C) Supernatants containing the indicated ECD proteins (mouse, long isoform) were diluted 1:2 into the indicated buffers and analyzed by SDS-PAGE and Western blot under reducing or non reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( D ) Phoenix expressing indicated were lysed in 1 % SDS in PBS or 10mM MES pH 6 . 1 buffers lysates were analyzed by SDS - PAGE Western blot under reducing conditions . Anti - DNGR - 1 antibody ( clone 397 ) followed by HRP - conjugated secondary anti - rat antibody was used for detection . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(D) Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10mM MES pH 6.1 buffers and the lysates were analyzed by SDS-PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) Decreasing amounts of F - actin were spotted onto a nitrocellulose binding of indicated DNGR - 1 ECD ( mouse , long isoform ) was tested in PBS or MES pH 6 . 1 buffers . HRP - conjugated anti - FLAG antibody was used for detection . signal was quantified by densitometry quantitation of four independent experiments is shown .

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(A) Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and quantitation of four independent experiments is shown.


( B C ) B3Z - Syk reporter expressing indicated mutant or WT DNGR - 1 receptors ( mouse , long isoform ) were incubated with UV - irradiated 293T ( dead : reporter is indicated ) , or with plate - bound anti - DNGR - 1 antibody or medium alone at 37 ° C overnight . Activation of NFAT reporter was measured at end of incubation period . Data are plotted as mean ± SD of experimental duplicates . One representative of three experiments is shown .

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(B and C) B3Z-Syk reporter cells expressing indicated mutant or WT DNGR-1 receptors (mouse, long isoform) were incubated with UV-irradiated 293T cells (ratio dead:reporter cells is indicated), or with plate-bound anti-DNGR-1 antibody or medium alone at 37°C overnight. Activation of the NFAT reporter was measured at the end of the incubation period. Data are plotted as mean ± SD of experimental duplicates. One representative of three experiments is shown.


( D ) Phoenix expressing indicated mutant or WT DNGR - 1 ( mouse , long isoform ) were treated with F - actin , F - actin buffer , anti - DNGR - 1 antibody or isotype - matched antibody of irrelevant specificity for 45 minutes , fixed in PFA , surface - stained for DNGR - 1 analyzed by flow cytometry . One representative of three experiments is shown .

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(D) Phoenix cells expressing indicated mutant or WT DNGR-1 proteins (mouse, long isoform) were treated with F-actin, F-actin buffer, anti-DNGR-1 antibody or isotype-matched antibody of irrelevant specificity for 45 minutes, fixed in PFA, surface-stained for DNGR-1 and analyzed by flow cytometry. One representative of three experiments is shown.


( A ) DNGR - 1 KO MuTu DCs were transduced to express indicated DNGR - 1 , FACS - sorted expanded . Surface ( left panel ) total ( right panel ) DNGR - 1 expression was determined by flow cytometry .

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(A) DNGR-1 KO MuTu DCs were transduced to express indicated DNGR-1 proteins, FACS-sorted and expanded. Surface (left panel) and total (right panel) DNGR-1 expression was determined by flow cytometry.


( B C ) Ovalbumin - expressing mouse embryonic fibroblasts ( bm1 T OVA MEFs ) were UV - irradiated , left to undergo secondary necrosis incubated overnight with MuTu transduced with indicated WT or mutant DNGR - 1 or an empty - vector control ( dead : MuTu is indicated ) OVA - specific pre - activated OT - I CD8 + T - lymphocytes . amount of IFNγ accumulating in culture medium was assessed using ELISA . One representative of two ( B ) or four ( C ) experiments is shown .

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(B and C) Ovalbumin-expressing mouse embryonic fibroblasts (bm1 T OVA MEFs) were UV-irradiated, left to undergo secondary necrosis and incubated overnight with MuTu cells transduced with indicated WT or mutant DNGR-1 proteins or an empty-vector control (ratio dead cells:MuTu cells is indicated) and OVA-specific pre-activated OT-I CD8+ T-lymphocytes. The amount of IFNγ accumulating in the culture medium was assessed using ELISA. One representative of two (B) or four (C) experiments is shown.


( B ) Immunoblot showing CENP - AEA level at indicated time in RPE - 1 .

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(B) Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells.


( C ) Representative immunofluorescence showing CENP - A reloading at CENP - B - marked centromeres in DLD - 1 . dashed circles contour nuclei . Scale bar , 5 µm .

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(C) Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres in DLD-1 cells. White dashed circles contour nuclei. Scale bar, 5 µm.


( D ) of of showing centromeric CENP - A 24 hr or 48 hr after IAA WO . Each dot represents one experiment ( ~ 30 - 50 per condition per experiment ) , error bars represent standard deviation ( SD ) of 5 independent experiments .

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(D) Quantification of the percentage of cells showing centromeric CENP-A 24 hr or 48 hr after IAA WO. Each dot represents one experiment (~30-50 cells per condition per experiment), error bars represent standard deviation (SD) of 5 independent experiments.


( E ) of centromeric CENP - A normalized to non - treated level . Each dot represents one experiment , error bars represent SD . Unpaired t : * * * p = 0 . 0005 .

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(E) Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, error bars represent SD. Unpaired t test: ***p= 0.0005.


( F ) Stills of live imaging to follow de novo CENP - AEA reloading in RPE - 1 harboring endogenously tagged CENP - Bmcherry . were taken every 15 min . dashed circles contour nuclei prior / after mitosis during mitosis on bright field .

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(F) Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright field images.


( G ) Dot plot showing timing of CENP - AEA reloading after anaphase onset in indicated lines . Each dot represents one , error bars represent standard deviation .

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(G) Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, error bars represent standard deviation.


( B ) Full coverage plot of chromosome 17 centromeric array ( chr17 : 22 , 500 , 000 - 26 , 900 , 000 of hg38 assembly ) showing enrichment of CENP - A by CUT & RUN - seq in untreated ( NT ) IAA wash out ( WO ) sample . CENP - A is reloaded to cen17 _ 1 ( D17Z1 ) . A shift of CENP - A occupancy to cen17 _ 2 ( D17Z1B ) is not detected .

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(B) Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000-26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected.


( F ) Representative showing ectopic CENP - A on a chromosome spread after induction of CENP - A overexpression in DLD - 1 . Centromere position is marked using immunofluorescence for CENP - B . Yellow dashed lines contour representative chromosomes . Scale bar , 2µm . Schematic to right illustrate observed CENP - AmRFP - DHFR localization pattern on a chromosome . Right panel : Comparison of CENP - ADHFR level per pixel at centromere on chromosome arms following IAA DOX / TMP treatment . Each dot represents one centromere or a region on chromosome arms .

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(F) Representative images showing ectopic CENP-A on a chromosome spread after induction of CENP-A overexpression in DLD-1 cells. Centromere position is marked using immunofluorescence staining for CENP-B. Yellow dashed lines contour representative chromosomes. Scale bar, 2µm. Schematic to the right illustrate the observed CENP-AmRFP-DHFR localization pattern on a chromosome. Right panel: Comparison of CENP-ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX/TMP treatment. Each dot represents one centromere or a region on the chromosome arms.


( G ) Schematic illustration for experiments shown in H , I . ( H ) Representative of chromosome spreads after re - expression of endogenous CENP - AEA into a CENP - AmRFP - DHFR overexpression background subsequent removal of overexpression as illustrated in ( G ) . Antibody against CENP - C was used to score for presence of functional centromere . Scale bar , 5µm . ( I ) of indicated events after one CENP - AOFF / ON cycle compared to treatment shown in ( G ) .

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(G) Schematic illustration for the experiments shown in H, I. (H) Representative images of chromosome spreads after re-expression of endogenous CENP-AEA into a CENP-AmRFP-DHFR overexpression background and subsequent removal of the overexpression as illustrated in (G). Antibody against CENP-C was used to score for the presence of the functional centromere. Scale bar, 5µm. (I) Quantification of the indicated events after one CENP-AOFF/ON cycle compared to treatment shown in (G).


( A ) Schematic illustration of CENP - AOFF / ON cycle performed in experiments shown in B , C . ( B ) Representative of de novo CENP - A reloading in CENP - B wild - type ( + / + ) CENP - B knock - out ( - / - ) . with centromeric CENP - A are marked with a red dashed contour while a yellow contour lines mark without centromeric CENP - A . Scale bar , 5 µm . ( C ) of relative number of RPE - 1 with centromeric CENP - A in indicated conditions . Each dot represents one experiment with at least 20 per condition . Error bars represent standard error of mean ( ) from 3 independent experiments .

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(A) Schematic illustration of the CENP-AOFF/ON cycle performed in the experiments shown in B, C. (B) Representative images of de novo CENP-A reloading in CENP-B wild-type (+/+) and CENP-B knock-out (-/-) cells. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. Scale bar, 5 µm. (C) Quantification of relative number of RPE-1 cells with centromeric CENP-A in the indicated conditions. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard error of the mean (SEM) from 3 independent experiments.


( E ) Representative of DLD - 1 CENP - B ( + / + ) or ( - / - ) in late M phase following one CENP - AOFF / ON cycle . EdU was used to confirm successful wash - out of Palbociclib progressing through S - phase . Yellow dashed lines contour nucleus of in late M phase . Scale bar 5 µm . ( F ) of indicated events observed in late M phase in indicated lines . Error bars show from 4 independent experiments .

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(E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.


( C ) Representative IF - FISH of mitotic spreads in non - treated ( NT ) following a CENP - AOFF / ON cycle ( IAA WO ) . Chromosomes are contoured by a green ( X ) , magenta ( Y ) or ( other autosomes ) dashed . centromeres of chromosome X / Y are highlighted by a green / magenta arrow . Scale bar , 2 µm .

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(C) Representative IF-FISH images of mitotic spreads in non-treated cell (NT) and following a CENP-AOFF/ON cycle (IAA WO). Chromosomes are contoured by a green (X), magenta (Y) or white (other autosomes) dashed line. The centromeres of chromosome X/Y are highlighted by a green/magenta arrow. Scale bar, 2 µm.


( D ) of CENP - C ( as read - out of CENP - A ) presence at X ( green ) or Y ( magenta ) centromere in indicated lines . Each dot represents one experiment . Error bars represent SD from 3 independent experiments . Unpaired t , * * p = 0 . 0042 .

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(D) Quantification of CENP-C (as read-out of CENP-A) presence at the X (green) or Y (magenta) centromere in the indicated cell lines. Each dot represents one experiment. Error bars represent SD from 3 independent experiments. Unpaired t test, **p=0.0042.


( G ) Representative IF - FISH showing CENP - C localization on Y chromosome in growing under selective pressure ( G418 ) following a CENP - AOFF / ON cycle . Chromosomes are highlighted by a dashed contour . CENP - C was found at native centromere position ( top panel , blue ) , Y chromosome fused to another chromosome indicated by increased of chromosome absence of Chr - Y painting probe ( second panel , orange ) or CENP - C was found on a different location on Y chromosome did not overlap with Cen - Y ( lower panels , red ) . Scale bar , 2 µm . ( H ) of indicated events depicted in G . Each dot represents one experiment ( > 20 spreads for experiment ) . Error bars represent from 3 independent experiments . Unpaired t - , * p = 0 . 0137 0 . 039 .

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(G) Representative IF-FISH images showing CENP-C localization on the Y chromosome in cell growing under selective pressure (G418) following a CENP-AOFF/ON cycle. Chromosomes are highlighted by a white dashed contour line. CENP-C was found at the native centromere position (top panel, blue), the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr-Y painting probe staining (second panel, orange) or CENP-C was found on a different location on the Y chromosome and did not overlap with Cen-Y DNA (lower panels, red). Scale bar, 2 µm. (H) Quantification of indicated events depicted in G. Each dot represents one experiment (>20 spreads for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t-test, *p=0.0137 and 0.039.


( B ) Representative immunofluorescence showing CENP - C recruitment at LacO array in indicated conditions . LacO array is displayed in insets .

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(B) Representative immunofluorescence images showing CENP-C recruitment at the LacO array in the indicated conditions. LacO array is displayed in the insets.


( C ) Bar plot showing frequency of CENP - C ( orange ) or CENP - A ( green ) recruitment to LacO array in indicated conditions . Each dot represents one independent experiment ( > 20 analyzed for experiment ) .

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(C) Bar plot showing frequency of CENP-C (orange) or CENP-A (green) recruitment to LacO array in the indicated conditions. Each dot represents one independent experiment (>20 cell analyzed for experiment).


( D ) of CENP - C ( orange ) or CENP - A ( green ) at LacO array in indicated conditions normalized to intensities in non - treated conditions at mCherry - LacI - CENP - B - LacO array using CENP - A or CENP - C antibody . Each dot represents one independent experiment ( > 20 analyzed for experiment ) . Error bars represent from 3 independent experiments .

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(D) Quantification of CENP-C (orange) or CENP-A (green) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non-treated conditions at the mCherry-LacI-CENP-B-LacO array using CENP-A or CENP-C antibody. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments.


( E ) Representative immunofluorescence showing CENP - A recruitment to ectopic mCherry - LacI - CENP - B in indicated conditions . LacO array is shown in inset .

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(E) Representative immunofluorescence images showing CENP-A recruitment to ectopic mCherry-LacI-CENP-B in the indicated conditions. LacO array is shown in the inset.


( F ) of frequency of CENP - C recruitment to LacO arrays by different LacI constructs . Error bars represent from 5 independent experiments .

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(F) Quantification of the frequency of CENP-C recruitment to LacO arrays by different LacI constructs. Error bars represent SEM from 5 independent experiments.


( G ) CENP - B immunoblot following GST pulldown experiments using GST tagged CENP - C ( 1 - 727 ) as bait with indicated as preys .

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(G) CENP-B immunoblot following GST pulldown experiments using GST tagged CENP-C (1-727) as bait and with the indicated proteins as preys.


( C ) Representative immunofluorescence showing CENP - A CENP - C reloading in indicated conditions . with centromeric CENP - A / CENP - C are marked with a red dashed contour while a yellow contour lines mark without centromeric CENP - A . centromere position is marked using immunofluorescence for CENP - B . dashed lines contour DAPI stained nucleus of representative .

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(C) Representative immunofluorescence images showing CENP-A and CENP-C reloading in the indicated conditions. Cells with centromeric CENP-A/CENP-C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. The centromere position is marked using immunofluorescence staining for CENP-B. White dashed lines contour DAPI stained nucleus of representative cells.


( D ) Bar plot showing of with CENP - A CENP - C co - localization after a CENP - A / COFF / ON cycle in indicated conditions . Error bars show SD from 5 independent experiments .

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(D) Bar plot showing the percentage of cells with CENP-A and CENP-C co-localization after a CENP-A/COFF/ON cycle in the indicated conditions. Error bars show SD from 5 independent experiments.


( E ) Box plot of CUT & RUN qPCR for CENP - A ( green ) or CENP - C ( orange ) . Box plot shows median , 25th 75th percentiles , whiskers show minimum maximum values . Results from 3 independent experiments including combined data from qPCR reactions using alpha - satellite primers primers binding at centromere of chromosome 4 . Enrichment is measured relative to IgG control normalized to Alu repeats .

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(E) Box plot of CUT&RUN qPCR for CENP-A (green) or CENP-C (orange). Box plot shows median, 25th and 75th percentiles, whiskers show minimum and maximum values. Results from 3 independent experiments including combined data from qPCR reactions using alpha-satellite primers and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


( C ) Representative immunofluorescence showing exogenous CENP - AmRFP - DHFR reloading in presence ( control ) , but not in absence ( sample ) of endogenous CENP - A CENP - C . with centromeric CENP - A are marked with a red dashed contour while a yellow contour marks without centromeric CENP - A . ( D ) of relative number of showing centromeric eCENP - AmRFP - DHFR in indicated conditions . Error bars represent from 3 independent experiments .

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(C) Representative immunofluorescence images showing exogenous CENP-AmRFP-DHFR reloading in the presence (control), but not in the absence (sample) of endogenous CENP-A and CENP-C. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-A. (D) Quantification of relative number of cells showing centromeric eCENP-AmRFP-DHFR in the indicated conditions. Error bars represent SEM from 3 independent experiments.


( E ) Bar plot of CUT & RUN qPCR using CENP - A antibody primers binding at centromere of chromosome 4 . Enrichment is measured relative to IgG control normalized to Alu repeats .

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(E) Bar plot of CUT&RUN qPCR quantification using CENP-A antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.