Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( D ) RT - qPCRs showing the levels of nam5 / 6 / 7 lncRNA population in the double mutant rrp6 ∆ dcr1 ∆ cells , relative to the single mutant rrp6 ∆ and dcr1 ∆ cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( D , E ) . P value was calculated using a two - tailed Student ' s t - 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

Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (D) RT-qPCRs showing the levels of nam5/6/7 lncRNA population in the double mutant rrp6∆ dcr1∆ cells, relative to the single mutant rrp6∆ and dcr1∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test.


Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( E ) RNA - IPs showing that Rrp6 - Myc13 binds to nam5 / 6 / 7 lncRNAs in a Mmi1 - dependent manner . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( D , E ) . P value was calculated using a two - tailed Student ' s t - 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

Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (E) RNA-IPs showing that Rrp6-Myc13 binds to nam5/6/7 lncRNAs in a Mmi1-dependent manner. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test.


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( A ) ChIPs assessing the localization of Mmi1 to pericentromeric DNA in WT and clr4 ∆ cells .

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

Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (A) ChIPs assessing the localization of Mmi1 to pericentromeric DNA in WT and clr4∆ cells.


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( B ) ChIPs showing the localization of Mmi1 ( dashed line ) and H3K9me2 ( black line ) to the pericentromeric nam5 / 6 / 7 DNA regions during the progression of the cell cycle . Cell synchronization was achieved by using cdc25 - ts cells ( see experimental procedures for more details ) . Cell synchronization was monitored by measuring the percentage of cells with a septum ( right part ) .

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

Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (B) ChIPs showing the localization of Mmi1 (dashed line) and H3K9me2 (black line) to the pericentromeric nam5/6/7 DNA regions during the progression of the cell cycle. Cell synchronization was achieved by using cdc25-ts cells (see experimental procedures for more details). Cell synchronization was monitored by measuring the percentage of cells with a septum (right part).


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( C ) RT - qPCRs monitoring the levels of nam5 / 6 / 7 ( left part ) and dg ( middle part ) lncRNA populations during the cell cycle . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( A , B and C ) .

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Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (C) RT-qPCRs monitoring the levels of nam5/6/7 (left part) and dg (middle part) lncRNA populations during the cell cycle. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (A, B and C).


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( D ) RT - PCRs monitoring the accumulation of nam7 lncRNAs and nam7 - L read - through transcripts in G2 / M and G1 / S phases from synchronized cells as in ( B ) . Red arrow heads point to the expected PCR products . The other bands are non - specific PCR products . From the scheme : black arrows , primers used for the reverse transcriptions ; black lines and red numbers , regions amplified by PCR .

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Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (D) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts in G2/M and G1/S phases from synchronized cells as in (B). Red arrow heads point to the expected PCR products. The other bands are non-specific PCR products. From the scheme: black arrows, primers used for the reverse transcriptions; black lines and red numbers, regions amplified by PCR.


( a ) Left , cells were fixed and stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . Solid arrows indicate cells with active Src in adhesions ; dashed arrows show cells with active Src in puncta . Scale bars , 20 μm . Right , percentage of cells with active Src localizing to intracellular structures was quantified . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) .

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(a) Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue). Solid arrows indicate cells with active Src in adhesions; dashed arrows show cells with active Src in puncta. Scale bars, 20 μm. Right, percentage of cells with active Src localizing to intracellular structures was quantified. Data are presented as mean±s.d. and significance is P0.001 (n=3).


( b ) Cells were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Merged and higher - magnification images of the outlined areas are also shown . Solid and dashed arrows show Src localization to adhesions or to puncta , respectively . Scale bars , 20 μm .

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(b) Cells were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Merged and higher-magnification images of the outlined areas are also shown. Solid and dashed arrows show Src localization to adhesions or to puncta, respectively. Scale bars, 20 μm.


( c ) Lysates from cells were immunoblotted with anti - FAK , anti - Src , anti - Yes , anti - Fyn , anti - pTyr - 416 - Src and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(c) Lysates from cells were immunoblotted with anti-FAK, anti-Src, anti-Yes, anti-Fyn, anti-pTyr-416-Src and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Cells were fixed and stained for anti - pTyr - 416 - Src ( red ) and co - stained for anti - LC3B , anti - Atg7 or anti - Atg12 ( all green ) . Scale bars , 20 μm . ( b ) Higher - magnification images of the areas outlined in a . Solid arrows indicate co - localization in focal adhesions and dashed arrows show puncta in the cytoplasm .

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(a) Cells were fixed and stained for anti-pTyr-416-Src (red) and co-stained for anti-LC3B, anti-Atg7 or anti-Atg12 (all green). Scale bars, 20 μm. (b) Higher-magnification images of the areas outlined in a. Solid arrows indicate co-localization in focal adhesions and dashed arrows show puncta in the cytoplasm.


( c ) LC3B was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - LC3B antibodies .

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(c) LC3B was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-LC3B antibodies.


( d ) Cells were transfected with 40 nM scrambled , Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti - Atg5 , anti - Atg12 and anti - actin antibodies ( lower panels ) or fixed and stained for anti - pTyr - 416 - Src ( green ) , anti - paxillin ( red ) and with DAPI ( blue ) . Dashed arrows point to intracellular phospho - Src - containing puncta ; solid arrows point to peripheral phospho - Src . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(d) Cells were transfected with 40 nM scrambled, Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti-Atg5, anti-Atg12 and anti-actin antibodies (lower panels) or fixed and stained for anti-pTyr-416-Src (green), anti-paxillin (red) and with DAPI (blue). Dashed arrows point to intracellular phospho-Src-containing puncta; solid arrows point to peripheral phospho-Src. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , cells were transiently transfected with an RFP - GFP tandem fluorescent - tagged LC3 ( RFP - GFP - LC3 ) . Scale bars , 20 μm . Bottom , the number of yellow puncta and the number of RFP LC3 - positive puncta in the merged images were counted and the total number of puncta per cell was calculated . Data are presented as mean ± s . d . ( n = 3 ) .

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(a) Top, cells were transiently transfected with an RFP-GFP tandem fluorescent-tagged LC3 (RFP-GFP-LC3). Scale bars, 20 μm. Bottom, the number of yellow puncta and the number of RFP LC3-positive puncta in the merged images were counted and the total number of puncta per cell was calculated. Data are presented as mean±s.d. (n=3).


b ) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti - pTyr - 416 - Src , anti - Src , anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown .

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b) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti-pTyr-416-Src, anti-Src, anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.


( c ) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti - pTyr - 416 - Src , anti - Src , anti - p53 and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(c) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti-pTyr-416-Src, anti-Src, anti-p53 and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , FAK − / − cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti - Src antibody ( green ) . Solid arrows indicate Src at the cell periphery ; dashed arrows indicate Src in autophagosomes . Scale bars , 20 μm . Bottom , quantification of dasatinib - treated cells with Src in puncta . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . Middle , lysates from cells treated with dasatinib were immunoblotted with anti - Src and anti - pTyr - 416 - Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown .

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

(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.


( b ) Top , FAK − / − cells were transiently transfected with SrcY527F - GFP or with Src - 251 - GFP ( green ) and stained for anti - LC3B ( red ) . Scale bars , 20 μm . Bottom , quantification of cells with co - localization between Src and LC3B . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) .

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(b) Top, FAK−/− cells were transiently transfected with SrcY527F-GFP or with Src-251-GFP (green) and stained for anti-LC3B (red). Scale bars, 20 μm. Bottom, quantification of cells with co-localization between Src and LC3B. Data are presented as mean±s.d. and significance is P0.001 (n=3).


( c ) Left , FAK − / − cells stably re - expressing wild - type FAK , FAKY397F or FAKY4F - Y9F were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate active Src in autophagosomes . Scale bars , 20 μm . Right , lysates from these cells were also immunoblotted with anti - FAK and anti - actin antibodies .

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(c) Left, FAK−/− cells stably re-expressing wild-type FAK, FAKY397F or FAKY4F-Y9F were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate active Src in autophagosomes. Scale bars, 20 μm. Right, lysates from these cells were also immunoblotted with anti-FAK and anti-actin antibodies.


( d ) Left , wild - type FAK , FAKY397F or FAKY4F - Y9F cells were transfected with RFP - GFP - LC3 . Solid arrows indicate co - localization and dotted arrows indicate its absence . Scale bars , 20 μm .

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(d) Left, wild-type FAK, FAKY397F or FAKY4F-Y9F cells were transfected with RFP-GFP-LC3. Solid arrows indicate co-localization and dotted arrows indicate its absence. Scale bars, 20 μm.


( e ) Lysates were immunoblotted with anti - LC3B and actin antibodies in the absence and presence of chloroquine . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(e) Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Wild - type FAK cells were suspended in 1 . 4 % methylcellulose solution in growth media and plated on agarose for 3 days . Top and bottom right , cells were recovered and cytospins prepared that were stained for anti - pTyr - 416 - Src ( red ) , LC3B ( green ) and with DAPI ( blue ) or for paxillin ( red ) , LC3B ( green ) and with DAPI ( blue ) . Higher - magnification images of the outlined areas are also shown . Solid arrows indicate localization of pTyr - 416 - Src in LC3B - positive puncta . Scale bars , 20 μm . Bottom left , immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti - pTyr - 397 FAK , anti - FAK , anti - pTyr - 416 and anti - Src antibodies .

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(a) Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies.


( b ) FAK + / + cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . A higher - magnification image of the area outlined is also shown . Scale bar , 20 μm .

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(b) FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm.


( c ) LC3B was immunoprecipitated ( IP ) from adherent or suspended FAK + / + and then immunoblotted with anti - Src and anti - LC3B antibodies . Lysates were also immunoblotted with anti - pTyr - 397 - FAK and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(c) LC3B was immunoprecipitated (IP) from adherent or suspended FAK+/+ and then immunoblotted with anti-Src and anti-LC3B antibodies. Lysates were also immunoblotted with anti-pTyr-397-FAK and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , FAK − / − cells were sparsely plated and then treated with 3 - MA ( for 48 h ) , chloroquine or dasatinib ( both for 24 h ) . Cells were then left at 37 ° C for 1 week to allow colony formation , fixed and then stained with crystal violet . Bottom , the number of colonies formed was then quantified and normalized against untreated controls . Data are presented as mean ± s . d . and significance is P0 . 001 for 3 - MA , P0 . 01 for chloroquine and P > 0 . 5 for dasatinib ( n = 3 ) .

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(a) Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h). Cells were then left at 37 °C for 1 week to allow colony formation, fixed and then stained with crystal violet. Bottom, the number of colonies formed was then quantified and normalized against untreated controls. Data are presented as mean±s.d. and significance is P0.001 for 3-MA, P0.01 for chloroquine and P>0.5 for dasatinib (n=3).


( b ) Top , wild - type FAK and FAK − / − cells were transiently transfected with two individual Atg5 shRNAs ( 1 and 2 ) , selected in puromycin and then the clonogenic assay was carried out as described above . Bottom , quantification ; data are presented as mean ± s . d . and significance is P > 0 . 1 for Atg5 A and B in wild - type FAK cells and P0 . 01 for Atg5 A and P0 . 05 for Atg5 B in FAK − / − cells ( n = 3 ) .

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(b) Top, wild-type FAK and FAK−/− cells were transiently transfected with two individual Atg5 shRNAs (1 and 2), selected in puromycin and then the clonogenic assay was carried out as described above. Bottom, quantification; data are presented as mean±s.d. and significance is P>0.1 for Atg5 A and B in wild-type FAK cells and P0.01 for Atg5 A and P0.05 for Atg5 B in FAK−/− cells (n=3).


( c ) Cells were also stained with TUNEL . The positive control was DNase1 - treated cells and the negative control lacked TUNEL reaction mix . Quantification is shown and data are presented as mean ± s . d . and significance is P0 . 005 for 3 - MA and P0 . 001 for Atg5 siRNA ( n = 3 ) .

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(c) Cells were also stained with TUNEL. The positive control was DNase1-treated cells and the negative control lacked TUNEL reaction mix. Quantification is shown and data are presented as mean±s.d. and significance is P0.005 for 3-MA and P0.001 for Atg5 siRNA (n=3).


( d ) FAK − / − cells were untreated or treated with dasatinib , 3 - MA , chloroquine or bafilomycin A ( BFA ) for 24 h , or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti - PARP antibody . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(d) FAK−/− cells were untreated or treated with dasatinib, 3-MA, chloroquine or bafilomycin A (BFA) for 24 h, or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti-PARP antibody. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Cells untreated , or treated with chloroquine for 24 h , were fixed and stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate co - localization in autophagosomes . Scale bars , 20 μm .

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(a) Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate co-localization in autophagosomes. Scale bars, 20 μm.


( b ) c - Cbl was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - c - Cbl antibodies .

(b) c-Cbl was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-c-Cbl antibodies.


( c , d ) Cells were transfected with scrambled siRNA or with c - Cbl siRNA for 72 h and then immunoblotted with anti - c - Cbl and actin antibodies ( c ) or stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ; d , left ) . Dashed arrows indicate co - localization and solid arrows indicate Src at focal adhesions . Scale bars , 20 μm . Quantification is shown ( d , right ) . Data are presented as mean ± s . d . and significance is P > 0 . 5 for FAK + / + cells and P0 . 001 for FAK − / − cells ( n = 3 ) .

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(c,d) Cells were transfected with scrambled siRNA or with c-Cbl siRNA for 72 h and then immunoblotted with anti-c-Cbl and actin antibodies (c) or stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green; d, left). Dashed arrows indicate co-localization and solid arrows indicate Src at focal adhesions. Scale bars, 20 μm. Quantification is shown (d, right). Data are presented as mean±s.d. and significance is P>0.5 for FAK+/+ cells and P0.001 for FAK−/− cells (n=3).


( e ) LC3B was immunoprecipitated from FAK − / − cells expressing either scrambled or c - Cbl siRNA and then immunoblotted with anti - Src and anti - LC3B antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

(e) LC3B was immunoprecipitated from FAK−/− cells expressing either scrambled or c-Cbl siRNA and then immunoblotted with anti-Src and anti-LC3B antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA resistant , HA - tagged wild - type c - Cbl or c - Cbl mutants with defective E3 ligase activity ( c - CblC381A or c - Cbl - 70Z ) . Left , cells were stained for anti - p - Tyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows show co - localization in intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P > 0 . 5 ( n = 3 ) .

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(a) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA resistant, HA-tagged wild-type c-Cbl or c-Cbl mutants with defective E3 ligase activity (c-CblC381A or c-Cbl-70Z). Left, cells were stained for anti-p-Tyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows show co-localization in intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P>0.5 (n=3).


( c ) GST - pulldown assay of HA - tagged wild - type c - Cbl , c - CblAA ( W802A , L805A ) and c - CblAAAA ( W802A , L803A , S804A , L805A ) expressed in HEK293T cells was carried out using GST - LC3 .

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(c) GST-pulldown assay of HA-tagged wild-type c-Cbl, c-CblAA (W802A, L805A) and c-CblAAAA (W802A, L803A, S804A, L805A) expressed in HEK293T cells was carried out using GST-LC3.


( d ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA - resistant HA - tagged wild - type c - Cbl or c - CblAAAA . Left , cells were stained for anti - pTyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows indicate pTyr - 416 localization to adhesions and dashed arrows indicate localization to intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P0 . 01 ( n = 3 ) . Uncropped images of blots are shown in Supplementary Fig . S9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA-resistant HA-tagged wild-type c-Cbl or c-CblAAAA. Left, cells were stained for anti-pTyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows indicate pTyr-416 localization to adhesions and dashed arrows indicate localization to intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P0.01 (n=3). Uncropped images of blots are shown in Supplementary Fig. S9.


A Gene targeting strategy . Left : max genomic locus , targeting construct and max targeted locus . In vitro Flipase ( Flip ) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 ( coding for bHLHLZip domain ) flanked by two loxP sites ( fl , flox allele ) . Arrows represent PCR primers used for genotyping MaxF9 and MaxF10 . Right : genomic PCR analysis of tail DNA from F1 mice . PCR products of max wt ( 430 bp ) and max flox ( 526 bp ) alleles . M , 1kb ladder .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O 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 Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.


Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) and spleen . Single - cell suspensions were prepared from BM or spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - ) , immature B cells ( Ly - 6c - CD49b - B220loIgM + CD43 - ) , and mature B cells ( Ly - 6c - CD49b - B220hiIgM + CD43 - ) To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) and spleen. Single-cell suspensions were prepared from BM or spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-), immature B cells (Ly-6c-CD49b-B220loIgM+CD43-), and mature B cells (Ly-6c-CD49b-B220hiIgM+CD43-) To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


Flow cytometry analyses of B cell populations ( GFP + ) in Bone Marrow ( BM ) Single - cell suspensions were prepared from BM of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . For BM analysis , cells were isolated from femora and tibiae . Pro - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B cells ( Ly - 6c - CD49b - B220 + IgM - CD43 - )

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Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) Single-cell suspensions were prepared from BM of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-)


Flow cytometry analyses of B cell populations ( GFP + ) in spleen . Single - cell suspensions were prepared from spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against the indicated markers , and analyzed by flow cytometry . spleen follicular B cells ( B220 + CD23 + CD21int ) , marginal zone B cells ( B220 + CD23 - CD21hi ) and immature ( newly formed / transitional stage 1 ) B cells ( B220 + CD23 - CD21lo ) . To identify KO cells ( GFP + ) all the plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in spleen. Single-cell suspensions were prepared from spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. spleen follicular B cells (B220+CD23+CD21int), marginal zone B cells (B220+CD23-CD21hi) and immature (newly formed/transitional stage 1) B cells (B220+CD23-CD21lo). To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


F , G , H Absolute numbers of B lymphocytes in Bone Marrow ( F , G ) and spleen ( F , H ) from MaxKO - mb1 ( n = 6 ) , MycKO - mb - 1 ( n = 3 ) , DKO - mb1 ( n = 5 ) and control HET - mb1 ( n = 4 ) mice . Columns represent mean values and error bars standard deviations . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments .

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F, G, H Absolute numbers of B lymphocytes in Bone Marrow (F, G) and spleen (F, H) from MaxKO-mb1 (n=6), MycKO-mb-1 (n=3), DKO-mb1 (n=5) and control HET-mb1 (n=4) mice. Columns represent mean values and error bars standard deviations. *p<0.05, **p<0.01, ***p<0.001. The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments.


I Cell cycle analysis by flow cytometry of sorted GFP + pre - and pro - B ( B220 + IgM - ) , immature ( B220loIgM + ) and mature ( B220hiIgM + ) bone marrow B cells from MaxKO - mb1 , MycKO - mb1 , DKO - mb1 and HET - mb1 control mice . Mice ( n = 5 ) were injected ( i . p . ) with 200 µg of EdU and analyzed 4 h later . Single - cell suspensions were prepared , stained and sorted . Sorted cells were subsequently stained with propidium iodide for DNA content . Experiment representative of at least three independent experiments . Dotted line is shown for EdU MFI comparison .

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I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.


J Percentage of annexin - V + GFP + cells in the BM of MaxKO - mb1 ( n = 5 ) , MycKO - mb1 ( n = 3 ) , DKO - mb1 ( n = 3 ) and control ( n = 4 ) mice . Pro - B , pre - B , immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E . * p < 0 . 05 , * * * p < 0 . 001 . Experiment representative of at least three independent experiments .

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J Percentage of annexin-V+GFP+ cells in the BM of MaxKO-mb1(n=5), MycKO-mb1(n=3), DKO-mb1 (n=3) and control (n=4) mice. Pro-B, pre-B, immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E. *p<0.05, ***p<0.001. Experiment representative of at least three independent experiments.


Flow cytometry analysis and absolute numbers of B220loCD138 + ( plasmablasts ) ( A , B ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , and DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted and activated in vitro with LPS plus IL - 4 for 72 h . Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values and error bars standard deviations . Experiment representative of at least three independent experiments .

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Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


Flow cytometry analysis and absolute numbers of B220 + IgG1 + switched B cells ( C , D ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , and DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted and activated in vitro with LPS plus IL - 4 for 72 h . Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values and error bars standard deviations . Experiment representative of at least three independent experiments .

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Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


E Spleen B220 + GFP + B lymphocytes were stained with 7AAD and annexin - V to monitor apoptosis . 7 - AAD - annexin - v - ( alive ) , 7 - AAD - annexin - v + ( early apoptotic ) , 7 - AAD + annexin - v + ( late apoptotic ) , 7 - AAD + annexin - v - ( necrosis ) . F Relative numbers of live , early apoptotic , late apoptotic and necrotic cells from E .

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E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E.


G CD69 surface expression in purified B220 + GFP + mature spleen cells . Sorted B cells were activated in vitro with LPS plus IL - 4 for 72 h , stained and analyzed by flow cytometry . The solid grey peak represents the expression levels of CD69 marker in non - activated mature B lymphocytes at day 0 . H Relative numbers of CD69 + B lymphocytes in the cultures from G ( n = 4 ) . Experiment representative of at least three independent experiments .

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G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments.


I Analysis of germinal center ( GC ) formation in the spleen of MaxKO - cd19 , Myc - KO - cd19 , DKO - cd19 and heterozygous control mice immunized with TNP - KLH . Representative images of frozen spleen sections stained with IgM ( grey / blue ) , PNA ( GC marker ; red ) , and GFP ( Max - , c - Myc - or c - Myc / Max - deficient B cells ; green ) . Scale bar , 80µm .

I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.


J Upper , Number of GCs per mouse in spleen sections of immunized MaxKO - cd19 ( n = 3 mice , n = 186 follicles analyzed ) , MycKO - cd19 ( n = 3 mice , n = 178 follicles analyzed ) , DKO - cd19 ( n = 3 mice , n = 191 follicles analyzed ) and heterozygous control mice ( n = 3 mice , n = 203 follicles analyzed ) . Lower , Frequencies of GC ( PNA + ) GFP + ( deleted cells ) or GFP - ( non - deleted cells ) in immunized MaxKO - cd19 ( n = 13 GCs ) , MycKO - cd19 ( n = 21 GCs ) , DKO - cd19 ( n = 27 GCs ) and heterozygous control ( n = 62 GCs ) mice . Analysis was performed at 13 days post - immunization . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


A Number of cell divisions in activated mature B lymphocytes . Sorted B220 + GFP + spleen cells from HET - cd19 ( n = 5 ) , MaxKO - cd19 ( n = 5 ) , MycKO - cd19 ( n = 3 ) , and DKO - cd19 ( n = 3 ) mice were stained with a cell tracer , activated in vitro with LPS plus IL - 4 for 72 h and analyzed by flow cytometry . Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar , 500 µm . 4 × magnification . D Forward scatter ( FSC ) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison .

A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar, 500 µm. 4× magnification. D Forward scatter (FSC) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison.


Cell cycle analysis of sorted B220 + GFP + B lymphocytes activated in vitro . Cells were activated and EdU was added 4 h before harvesting . Cells were stained with propidium iodide to measure DNA content . Experiment representative of at least three independent experiments . An arbitrary dashed line shows the difference of EdU MFI . S phase mean fluorescence intensity ( MFI ) : 7871 HET , 6512 MaxKO , 5860 MycKO , 6420

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Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKO


Representation of labeling with nucleotide analogues CldU ( red ) and IdU ( green ) and photographs of DNA fibers from sorted B220 + GFP + B lymphocytes activated for 48 h . Scale bar , 10 µm .

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Representation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated for 48 h. Scale bar, 10 µm.


H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments . Horizontal bars represent median values . 150 - 250 forks were measured per condition . * * * , p < 0 . 001 ; * * , p < 0 .

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H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments. Horizontal bars represent median values. 150-250 forks were measured per condition. ***, p<0.001; **, p<0.01


I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220 + GFP + spleen cells were activated in vitro with LPS plus IL - 4 for 72 h . MEK2 , SMC1 are shown as loading controls . The blots shown are representative of three independent experiments .

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I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.


A Bar plot showing the number of DEGs ( adj . p - value < 0 . 01 , FC + 1 . 5 in red , 1 . 5 < FC > - 1 . 5 in white , FC < - 1 . 5 in blue ) . Sorted B220 + GFP + spleen B cells from MaxKO - cd19 , MycKO - cd19 , DKO - cd19 and control HET - cd19 mice were activated with LPS plus IL - 4 for 72 h and analyzed . Two samples for each condition were used .

A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.


C Hierarchical clustering heat map of DEGs in any comparison ( two samples per condition ) . Dark blue in left panel indicates significance for that contrast column . Colour code : red represents upregulation , white , unchanged , and blue downregulation of expression . Gender : colour code for genetic condition . Pink are female and blue colour male mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Hierarchical clustering heat map of DEGs in any comparison (two samples per condition). Dark blue in left panel indicates significance for that contrast column. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. Gender: colour code for genetic condition. Pink are female and blue colour male mice.


D Venn diagrams of up - ( left ) and down - regulated genes ( right ) for all three conditions ( + / - 1 . 5 - FC and p < 0 . 01 ) . Heatmaps of genes only deregulated in MaxKO - cd19 , MycKO - cd19 , or DKO - cd19 mice are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Venn diagrams of up- (left) and down-regulated genes (right) for all three conditions (+/-1.5-FC and p<0.01). Heatmaps of genes only deregulated in MaxKO-cd19, MycKO-cd19, or DKO-cd19 mice are shown.


B Heatmaps of genes involved in the relevant pathways Colour code : red represents upregulation , white , unchanged , and blue downregulation of expression .

O O 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 Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.


C qPCR analysis of up - and down - regulated genes Pooled RNA was prepared from sorted B220 + GFP + spleen B cells of MaxKO - cd19 ( n = 4 ) , MycKO - cd19 ( n = 4 ) , or DKO - cd19 ( n = 4 ) and control HET - cd19 ( n = 4 ) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72 , med13l , card11 , ssh2 , cdc7 , mrpl12 , rpl39 , and cysc . max and c - myc were included as controls and actin was used for normalization .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.


A Double immunofluorescence for GFP ( green ) and the BP marker TBR2 ( yellow ) , combined with DAPI staining ( white ) , of a 57 days - old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and TBR2 + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells that are TBR2 + in 57 days - old chimpanzee cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 9 control and 9 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * * , P < 0 . 001 ( two - sided Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and the BP marker TBR2 (yellow), combined with DAPI staining (white), of a 57 days-old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells that are TBR2+ in 57 days-old chimpanzee cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 9 control and 9 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).


C Triple immunofluorescence for GFP ( green ) , the cycling cell marker Ki67 ( magenta ) and TBR2 ( yellow ) , combined with DAPI staining ( white ) , of a 59 days - old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + , Ki67 + and TBR2 + triple - positive cells . Scale bar , 50 µ D Quantification of the proportion of GFP + cells in the SVZ / NL that are Ki67 + and TBR2 + double - positive in 59 days - old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 8 control and 8 ARHGAP11B - transfected cerebral organoids of four independent batches each ; error bars indicate SD ; * , P < 0 . 05 ( one - sided Wilcoxon rank sum 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Triple immunofluorescence for GFP (green), the cycling cell marker Ki67 (magenta) and TBR2 (yellow), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+, Ki67+ and TBR2+ triple-positive cells. Scale bar, 50 µ D Quantification of the proportion of GFP+ cells in the SVZ/NL that are Ki67+ and TBR2+ double-positive in 59 days-old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 8 control and 8 ARHGAP11B-transfected cerebral organoids of four independent batches each; error bars indicate SD; *, P <0.05 (one-sided Wilcoxon rank sum test).


A Double immunofluorescence for GFP ( green ) and the radial glia marker HOPX ( yellow ) , combined with DAPI staining ( white ) , of a 59 days - old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and HOPX + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells in the SVZ / NL that are HOPX + in 59 days - old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 10 control and 10 ARHGAP11B - transfected cerebral organoids of three independent batches each ; error bars indicate SD ; * * , P < 0 . 01 ( one - sided Wilcoxon rank sum test ) . C Double immunofluorescence for GFP ( green ) and HOPX ( yellow ) , combined with DAPI staining ( white ) , of a 61 days - old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and HOPX + double - positive cells . Scale bar , 50 µm . D Quantification of the proportion of GFP + cells in the SVZ / NL that are HOPX + in 61 days - old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 5 control and 7 ARHGAP11B - transfected cerebral organoids of three independent batches each ; error bars indicate SD ; * , P < 0 . 05 ( two - sided Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and the radial glia marker HOPX (yellow), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells in the SVZ/NL that are HOPX+ in 59 days-old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids of three independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test). C Double immunofluorescence for GFP (green) and HOPX (yellow), combined with DAPI staining (white), of a 61 days-old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and HOPX+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL that are HOPX+ in 61 days-old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 5 control and 7 ARHGAP11B-transfected cerebral organoids of three independent batches each; error bars indicate SD; *, P<0.05 (two-sided Student's t-test).


A Double immunofluorescence for GFP ( green ) and the neuron marker Hu ( magenta ) , combined with DAPI staining ( white ) , of a 61 days - old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and Hu + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells that are Hu + in 61 days - old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 7 control and 11 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * , P < 0 . 01 ( two - sided Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and the neuron marker Hu (magenta), combined with DAPI staining (white), of a 61 days-old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and Hu+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells that are Hu+ in 61 days-old chimpanzee cerebral organoids 10 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 7 control and 11 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; **, P <0.01 (two-sided Student's t-test).


C Double immunofluorescence for GFP ( green ) and the deep - layer neuron marker CTIP2 ( magenta ) , combined with DAPI staining ( white ) , of a 59 days - old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and CTIP2 + double - positive cells . Scale bar , 50 µm . D Quantification of the proportion of GFP + cells in the SVZ / NL that are CTIP2 + in 59 - days old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 10 control and 10 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * * , P < 0 . 001 ( two - sided Student ' s t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and the deep-layer neuron marker CTIP2 (magenta), combined with DAPI staining (white), of a 59 days-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and CTIP2+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL that are CTIP2+ in 59-days old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 10 control and 10 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; ***, P <0.001 (two-sided Student's t-test).


E Double immunofluorescence for GFP ( green ) and the upper - layer neuron marker SATB2 ( magenta ) , combined with DAPI staining ( white ) , of a 66 days - old chimpanzee cerebral organoid 15 days after electroporation with GFP expression plasmid plus either control plasmid ( top ) or ARHGAP11B expression plasmid ( bottom ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and SATB2 + double - positive cells . Scale bar , 50 µm . F Quantification of the proportion of GFP + cells in the SVZ / NL that are SATB2 + in 66 days - old chimpanzee cerebral organoids 15 days after electroporation with GFP expression plasmid plus either control plasmid ( dark red ) or ARHGAP11B expression plasmid ( light red ) . Data are the mean of 6 control and 4 ARHGAP11B - transfected cerebral organoids of two independent batches each ; error bars indicate SD ; * * , P < 0 . 01 ( one - sided Wilcoxon rank sum test ) .

E Double immunofluorescence for GFP (green) and the upper-layer neuron marker SATB2 (magenta), combined with DAPI staining (white), of a 66 days-old chimpanzee cerebral organoid 15 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and SATB2+ double-positive cells. Scale bar, 50 µm. F Quantification of the proportion of GFP+ cells in the SVZ/NL that are SATB2+ in 66 days-old chimpanzee cerebral organoids 15 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of 6 control and 4 ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; **, P <0.01 (one-sided Wilcoxon rank sum test).


A Double immunofluorescence for GFP ( green ) and the thymidine analog BrdU ( magenta , combined with DAPI staining ( white ) , of 57 days - old human ( top panels ) and chimpanzee ( bottom panels ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( upper panels ) or ARHGAP11A220 expression plasmid ( lower panels ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and BrdU + double - positive cells . Scale bar , 50 µm . B Quantification of the proportion of GFP + cells in the SVZ / NL that are BrdU + in 57 days - old human ( blue ) and chimpanzee ( red ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( dark blue and dark red ) or ARHGAP11A220 expression plasmid ( light blue and light red ) . Data are the mean of 12 human control , 12 human ARHGAP11A220 - transfected , 9 chimpanzee control and 8 chimpanzee ARHGAP11A220 - transfected cerebral organoids ( grown from one human and one chimpanzee iPSC line , respectively ) of six ( human ) and four ( chimpanzee ) independent batches each ; error bars indicate SD ; n . s . , not significant ; * * , P < 0 . 01 ( Kruskal Wallis 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and the thymidine analog BrdU (magenta, combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and BrdU+ double-positive cells. Scale bar, 50 µm. B Quantification of the proportion of GFP+ cells in the SVZ/NL that are BrdU+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 12 human control, 12 human ARHGAP11A220-transfected, 9 chimpanzee control and 8 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of six (human) and four (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; **, P <0.01 (Kruskal Wallis test).


C Double immunofluorescence for GFP ( green ) and TBR2 ( yellow ) , combined with DAPI staining ( white ) , of 57 days - old human ( top panels ) and chimpanzee ( bottom panels ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( upper panels ) or ARHGAP11A220 expression plasmid ( lower panels ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and TBR2 + double - positive cells . Scale bar , 50 µm . D Quantification of the proportion of GFP + cells in the SVZ / NL that are TBR2 + in 57 days - old human ( blue ) and chimpanzee ( red ) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid ( dark blue and dark red ) or ARHGAP11A220 expression plasmid ( light blue and light red ) . Data are the mean of 9 human control , 9 human ARHGAP11A220 - transfected , 10 chimpanzee control and 10 chimpanzee ARHGAP11A220 - transfected cerebral organoids ( grown from one human and one chimpanzee iPSC line , respectively ) of four ( human ) and three ( chimpanzee ) independent batches each ; error bars indicate SD ; n . s . , not significant ; * , P < 0 . 05 ( Kruskal Wallis 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and TBR2 (yellow), combined with DAPI staining (white), of 57 days-old human (top panels) and chimpanzee (bottom panels) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (upper panels) or ARHGAP11A220 expression plasmid (lower panels). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bar, 50 µm. D Quantification of the proportion of GFP+ cells in the SVZ/NL that are TBR2+ in 57 days-old human (blue) and chimpanzee (red) cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark blue and dark red) or ARHGAP11A220 expression plasmid (light blue and light red). Data are the mean of 9 human control, 9 human ARHGAP11A220-transfected, 10 chimpanzee control and 10 chimpanzee ARHGAP11A220-transfected cerebral organoids (grown from one human and one chimpanzee iPSC line, respectively) of four (human) and three (chimpanzee) independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05 (Kruskal Wallis test).


A Double immunofluorescence for GFP ( green ) and the bRG marker PTPRZ1 ( magenta ) , combined with DAPI staining ( white ) , of 60 days - old human forebrain organoids , generated from ARHGAP11A plus ARHGAP11B double - knockout clone 16 . Images were obtained 10 days after electroporation with GFP expression plasmid plus either ARHGAP11A plus ARHGAP11B expression plasmids ( ARHGAP11A + B - rescue , referred to as Control ) , ARHGAP11B expression plasmid only ( ARHGAP11B - rescue , referred to as ARHGAP11A - KO ) , or ARHGAP11A expression plasmid only ( ARHGAP11A - rescue , referred to as ARHGAP11B - KO ) . Tick marks indicate the borders of the VZ and SVZ / NL ; arrowheads indicate examples of GFP + and PTPRZ1 + double - positive cells . Scale bar , 50 µm B Quantification of the proportion of GFP + cells in the SVZ / NL that are PTPRZ1 + in 60 days - old ARHGAP11A plus ARHGAP11B double - knockout ( 11A + 11B KO ) human forebrain organoids . Organoids were generated from clone 16 ( left ) and clone j ( right ) , and data were obtained 10 days after the indicated electroporation with GFP expression plasmid plus either ARHGAP11A ( 11A ) plus ARHGAP11B ( 11B ) expression plasmids ( referred to as Control , as this constitutes an ARHGAP11A + B double - rescue in the 11A + 11B KO organoids ; dark blue ) , ARHGAP11B ( 11B ) expression plasmid only ( referred to as 11A - KO , as this constitutes a selective ARHGAP11B rescue in the 11A + 11B KO organoids ; light blue ) , or ARHGAP11A ( 11A ) expression plasmid only ( referred to as 11B - KO , as this constitues a selective ARHGAP11A rescue in the 11A + 11B KO organoids ; dark red ) . For clone 16 , data are the mean of 8 control , 8 11A - KO and 8 11B - KO human forebrain organoids of two independent batches each ; for clone j , data are the mean of 9 control , 10 11A - KO and 8 11B - KO human forebrain organoids of three independent batches each ; error bars indicate SD ; n . s . , not significant ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ( Kruskal Wallis 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Double immunofluorescence for GFP (green) and the bRG marker PTPRZ1 (magenta), combined with DAPI staining (white), of 60 days-old human forebrain organoids, generated from ARHGAP11A plus ARHGAP11B double-knockout clone 16. Images were obtained 10 days after electroporation with GFP expression plasmid plus either ARHGAP11A plus ARHGAP11B expression plasmids (ARHGAP11A+B-rescue, referred to as Control), ARHGAP11B expression plasmid only (ARHGAP11B-rescue, referred to as ARHGAP11A-KO), or ARHGAP11A expression plasmid only (ARHGAP11A-rescue, referred to as ARHGAP11B-KO). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and PTPRZ1+ double-positive cells. Scale bar, 50 µm B Quantification of the proportion of GFP+ cells in the SVZ/NL that are PTPRZ1+ in 60 days-old ARHGAP11A plus ARHGAP11B double-knockout (11A+11B KO) human forebrain organoids. Organoids were generated from clone 16 (left) and clone j (right), and data were obtained 10 days after the indicated electroporation with GFP expression plasmid plus either ARHGAP11A (11A) plus ARHGAP11B (11B) expression plasmids (referred to as Control, as this constitutes an ARHGAP11A+B double-rescue in the 11A+11B KO organoids; dark blue), ARHGAP11B (11B) expression plasmid only (referred to as 11A-KO, as this constitutes a selective ARHGAP11B rescue in the 11A+11B KO organoids; light blue), or ARHGAP11A (11A) expression plasmid only (referred to as 11B-KO, as this constitues a selective ARHGAP11A rescue in the 11A+11B KO organoids; dark red). For clone 16, data are the mean of 8 control, 8 11A-KO and 8 11B-KO human forebrain organoids of two independent batches each; for clone j, data are the mean of 9 control, 10 11A-KO and 8 11B-KO human forebrain organoids of three independent batches each; error bars indicate SD; n.s., not significant; *, P <0.05; **, P <0.01; ***, P <0.001 (Kruskal Wallis test)


B ) Representative CD3 immunohistochemistry staining of a muscle biopsy from a patient with inclusion body myositis ( 6 - IBM ) .

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B) Representative CD3 immunohistochemistry staining of a muscle biopsy from a patient with inclusion body myositis (6-IBM).


D ) UMAP feature plots displaying the normalized expression of CD4 , CD8A and CD8B genes . Data information : Each dot represents one cell .

D) UMAP feature plots displaying the normalized expression of CD4, CD8A and CD8B genes. Data information: Each dot represents one cell.


B ) UMAP feature plots displaying the normalized expression of CD4 , CD8A and CD8B genes . Data information : Each dot represents one cell .

B) UMAP feature plots displaying the normalized expression of CD4, CD8A and CD8B genes. Data information: Each dot represents one cell.


C ) Violin plots displaying the normalized expression level of selected genes per cluster in muscle T cells ( n = 6 IIM , n = 1 undefined myopathy ) . Data information : CM : central memory ; EM : effector memory ; TRM : tissue resident memory ; Tregs : regulatory T cells . IIM : Idiopathic Inflammatory Myopathies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Violin plots displaying the normalized expression level of selected genes per cluster in muscle T cells (n=6 IIM, n=1 undefined myopathy). Data information: CM: central memory; EM: effector memory; TRM: tissue resident memory; Tregs: regulatory T cells. IIM: Idiopathic Inflammatory Myopathies.


D ) Violin plots displaying the normalized expression level of selected genes per patient in the four TRM clusters ( n = 6 IIM , n = 1 undefined myopathy ) . IMNM : Immune - Mediated Necrotizing Myopathy ; DM : DermatoMyositis ; ASyS : AntiSYnthetase Syndrome ; IBM : Inclusion Body

D) Violin plots displaying the normalized expression level of selected genes per patient in the four TRM clusters (n=6 IIM, n=1 undefined myopathy). IMNM: Immune-Mediated Necrotizing Myopathy; DM: DermatoMyositis; ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body Myositis


E ) Representative immunofluorescence stainings of HOBIT ( red ) , CD3 ( green ) , dystrophin ( purple ) and Hoechst 33342 ( blue ) performed on muscle tissue for patient 6 ( IBM ) , patient 3 ( DM ) and 4 ( ASyS ) . Images were acquired using a LSM 880 confocal without Airyscan microscope ( 63x oil objective ) . Scale bar = 20μm . The white arrows show HOBIT + T cells . DM : DermatoMyositis ; ASyS : AntiSYnthetase Syndrome ; IBM : Inclusion Body

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence stainings of HOBIT (red), CD3 (green), dystrophin (purple) and Hoechst 33342 (blue) performed on muscle tissue for patient 6 (IBM), patient 3 (DM) and 4 (ASyS). Images were acquired using a LSM 880 confocal without Airyscan microscope (63x oil objective). Scale bar=20μm. The white arrows show HOBIT+ T cells. DM: DermatoMyositis; ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body Myositis


F ) Isotype mouse and rabbit IgG1 immunofluorescence stainings . IBM : Inclusion Body

F) Isotype mouse and rabbit IgG1 immunofluorescence stainings. IBM: Inclusion Body Myositis


G ) Quantification of HOBIT + T cells in muscle biopsies of patient 6 ( IBM ) , patient 16 ( IBM ) , patient 3 ( DM ) and 4 ( ASyS ) . The number of counted CD3 - positive T cells is indicated under each bar as n . ASyS : AntiSYnthetase Syndrome ; IBM : Inclusion Body

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G) Quantification of HOBIT+ T cells in muscle biopsies of patient 6 (IBM), patient 16 (IBM), patient 3 (DM) and 4 (ASyS). The number of counted CD3-positive T cells is indicated under each bar as n. ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body Myositis


A ) Stacked bar plots showing the frequency of T - cell clones per patient in the muscle ( left ) and peripheral blood ( PB ) ( right ) , including non - expanded ( unique , lightest shade ) and expanded clones ( colored from light to dark shades , from less expanded to more expanded clones ) ) . IMNM : Immune - Mediated Necrotizing Myopathy ; DM : DermatoMyositis ; ASyS : AntiSYnthetase Syndrome IBM : Inclusion Body Myositis ; IIM : Idiopathic Inflammatory Myopathies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stacked bar plots showing the frequency of T-cell clones per patient in the muscle (left) and peripheral blood (PB) (right), including non-expanded (unique, lightest shade) and expanded clones (colored from light to dark shades, from less expanded to more expanded clones)). IMNM: Immune-Mediated Necrotizing Myopathy; DM: DermatoMyositis; ASyS: AntiSYnthetase Syndrome IBM: Inclusion Body Myositis; IIM: Idiopathic Inflammatory Myopathies.


B ) Feature plots of IFI44L expression levels in each patient at early diagnosis in muscle T cells ( upper panel ) and peripheral blood memory T cells ( lower panel ) . Data information : IMNM : Immune - Mediated Necrotizing Myopathy ; DM : DermatoMyositis ASyS : AntiSYnthetase Syndrome ; IBM : Inclusion Body Myositis ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Feature plots of IFI44L expression levels in each patient at early diagnosis in muscle T cells (upper panel) and peripheral blood memory T cells (lower panel). Data information: IMNM: Immune-Mediated Necrotizing Myopathy; DM: DermatoMyositis ASyS: AntiSYnthetase Syndrome; IBM: Inclusion Body Myositis;


( A ) Pearson correlation between ZEB1 and MITF mRNA expression in 61 melanomacell lines available through the CCLE .

(A) Pearson correlation between ZEB1 and MITF mRNA expression in 61 melanomacell lines available through the CCLE.


( B ) ZEB1 , ZEB2 , TWIST1 and MITF expression in a panel of BRAFV600 - mutated melanomacells assessed by Western blot . GLO and C - 09 . 10cells are patient - derived short - term cultures . Actin was used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O 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) ZEB1, ZEB2, TWIST1 and MITF expression in a panel of BRAFV600-mutated melanomacells assessed by Western blot. GLO and C-09.10cells are patient-derived short-term cultures. Actin was used as a loading control.


( C ) Quantitative PCR analyses of ZEB1 , ZEB2 , TWIST1and MITF in the same panel of cell lines . mRNA expression levels are represented relatively to C - 09 . 10 cells , in which the levels were arbitrarily fixed at 1 ( n = 3 , mean ± SD ) . The dotted line separates ZEB1high ( left ) and ZEB1low ( right ) cell 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 O 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) Quantitative PCR analyses of ZEB1, ZEB2, TWIST1and MITF in the same panel of cell lines. mRNA expression levels are represented relatively to C-09.10 cells, in which the levels were arbitrarily fixed at 1 (n=3, mean ± SD). The dotted line separates ZEB1high (left) and ZEB1low (right) cell lines.


( D ) Tukey box plot of ZEB1 , ZEB2 , TWIST1 , and MITF mRNA expression according to the IC50 of the drug ( µM ) administered ( BRAFi / MEKi ) , in melanoma cell lines from the CCLE ( n = 28 ) ( Student ' s t - Test ) . High ZEB1 , low ZEB2 , and low MITF expression levels were correlated with BRAFi ( PLX4720 ) and MEKi ( AZD6244 ) resistance . PLX4720 is an analog of PLX4032 .

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

(D) Tukey box plot of ZEB1, ZEB2, TWIST1, and MITF mRNA expression according to the IC50 of the drug (µM) administered (BRAFi/MEKi), in melanoma cell lines from the CCLE (n=28) (Student's t-Test). High ZEB1, low ZEB2, and low MITF expression levels were correlated with BRAFi (PLX4720) and MEKi (AZD6244) resistance. PLX4720 is an analog of PLX4032.


( E ) IC50 values of PLX4032 ( µM ) in the panel of BRAFV600 melanoma cells as determined by ATP assay ( n = 3 , mean ± SD ) . For SKMEL24 and WM793 , IC50 was > 8µM .

O O O O O O O O O O O O O O O O O O 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) IC50 values of PLX4032 (µM) in the panel of BRAFV600 melanoma cells as determined by ATP assay (n=3, mean ± SD). For SKMEL24 and WM793, IC50 was >8µM.


( A ) Pearson correlation between ZEB1 and MITF mRNA expression levels in 467 melanomatumors from TCGA data set .

(A) Pearson correlation between ZEB1 and MITF mRNA expression levels in 467 melanomatumors from TCGA data set.


( B ) Representative pictures of ZEB1 and MITF immunostaining in primary melanomas . Scale bar = 40 µm . The aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype .

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(B) Representative pictures of ZEB1 and MITF immunostaining in primary melanomas. Scale bar = 40 µm. The aberrant activation of ZEB1 in melanomas is correlated with a MITFlow phenotype.


( C ) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from patients , classified in ZEB1 high , int and low subgroups , based on the intensity of ZEB1 staining and on the percentage of cells positive for ZEB1 . Scale bar = 80 µm . ( D ) Pie charts representing the distribution of ZEB1 alone ( upper part ) , or ZEB1 and TWIST1 ( lower part ) immunohistochemistry staining in tumors according to their initial response to vemurafenib + / - cobimetinib treatment . ZEB1 + / - TWIST1 levels are higher in MAPKi primary resistant melanomas ( initial non - responders ) compared to tumors that initially respond to treatment ( n = 70 , Fisher ' s exact test ) .

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(C) Representative pictures of ZEB1 immunostaining in BRAFV600 tumors from patients, classified in ZEB1 high, int and low subgroups, based on the intensity of ZEB1 staining and on the percentage of cells positive for ZEB1. Scale bar = 80 µm. (D) Pie charts representing the distribution of ZEB1 alone (upper part), or ZEB1 and TWIST1 (lower part) immunohistochemistry staining in tumors according to their initial response to vemurafenib +/- cobimetinib treatment. ZEB1 +/- TWIST1 levels are higher in MAPKi primary resistant melanomas (initial non-responders) compared to tumors that initially respond to treatment (n=70, Fisher's exact test).


( E ) Representative pictures of ZEB1 and MITF immunostainings , before and after vemurafenib treatment , in the tumor from patient 1 , exhibiting primary resistance to BRAFi . Scale bar = 80 µm . Right : Magnification of MITFhigh and MITFlow clones in the resistant tumor under treatment . Arrows indicate endothelial and stromal cells that also show positive staining for ZEB1 , besides tumor cells .

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(E) Representative pictures of ZEB1 and MITF immunostainings, before and after vemurafenib treatment, in the tumor from patient 1, exhibiting primary resistance to BRAFi. Scale bar = 80 µm. Right: Magnification of MITFhigh and MITFlow clones in the resistant tumor under treatment. Arrows indicate endothelial and stromal cells that also show positive staining for ZEB1, besides tumor cells.


( A ) PLX4032 IC50 ( µM ) of sensitive A375 and SKMEL5 and resistant ( A375 - R , SKMEL5 - R ) cell lines , as well as of GOKA and ESPcells , two BRAFi - resistant patient - derived short - term cultures , as determined by ATP assay ( n = 3 , mean ± SD ) . For ESP , IC50 was > 8µM .

(A) PLX4032 IC50 (µM) of sensitive A375 and SKMEL5 and resistant (A375-R, SKMEL5-R) cell lines, as well as of GOKA and ESPcells, two BRAFi-resistant patient-derived short-term cultures, as determined by ATP assay (n=3, mean ± SD). For ESP, IC50 was >8µM.


( B ) Western blot analyses of ZEB1 , TWIST1 and FRA1 in A375 - R and SKMEL5 - Rversus the parental naive cells , and in GOKA and ESP cells . GAPDH was used as a loading control .

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(B) Western blot analyses of ZEB1, TWIST1 and FRA1 in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. GAPDH was used as a loading control.


( C ) Quantitative PCR analyses of ZEB1and MITF in A375 - R and SKMEL5 - Rversus the parental naive cells , and in GOKA and ESP cells . mRNA expression levels are represented as arbitrary units ( a . u ) . Statistical difference relative to sensitive A375 cells is shown ( n = 3 , mean ± SD , Student ' s t - 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

(C) Quantitative PCR analyses of ZEB1and MITF in A375-R and SKMEL5-Rversus the parental naive cells, and in GOKA and ESP cells. mRNA expression levels are represented as arbitrary units (a.u). Statistical difference relative to sensitive A375 cells is shown (n=3, mean ± SD, Student's t-test).


( D ) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2 , 3 and 4 , before and after vemurafenib treatment . Scale bar = 40 µm . For ZEB1 staining in patient 4 , the inset shows a magnification . Arrows point at stromal cells ( s ) . All other cells positive for ZEB1 are tumor cells .

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

(D) Representative pictures of ZEB1 and MITF immunostainings in tumors from patients 2, 3 and 4, before and after vemurafenib treatment. Scale bar = 40 µm. For ZEB1 staining in patient 4, the inset shows a magnification. Arrows point at stromal cells (s). All other cells positive for ZEB1 are tumor cells.


( A ) A375 cells were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 and p75 . GAPDH was used as a loading control .

O O O O 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) A375 cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control.


( B ) Quantitative PCR analyses of MITF , p75 , ABCB5 and JARID1B upon ZEB1 expression . mRNA expression levels are represented relatively to control cells , in which the levels were fixed at 1 ( mean ± SD , n = 3 , Student ' s t - 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

(B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 expression. mRNA expression levels are represented relatively to control cells, in which the levels were fixed at 1 (mean ± SD, n=3, Student's t-test).


( C ) Soft agar colony formation assay upon ZEB1 expression . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - 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

(C) Soft agar colony formation assay upon ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).


( D ) 2 x 106 Control or ZEB1 - overexpressing A375 cells were injected subcutaneously into nude mice . The mean tumor volume for 5 mice is represented ± SEM ( Student ' s t - test ) .

(D) 2 x 106 Control or ZEB1-overexpressing A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented ± SEM (Student's t-test).


( E ) Western blot analyses of ZEB1 , MITF , P - ERK , and TWIST1 levels in control or ZEB1 - expressing cells + / - 150 nM PLX4032 treatment for 24 h . GAPDH was used as a loading control .

O O O O O O 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

(E) Western blot analyses of ZEB1, MITF, P-ERK, and TWIST1 levels in control or ZEB1-expressing cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.


( F ) FACS analyses of p75 cell surface expression upon ZEB1 overexpression , after 10 days treatment with or without 150 nM PLX4032 . Bar chart representing the mean percentage of p75 - high , int and low cells from 2 independent experiments ( Fisher ' s Exact Test ) .

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(F) FACS analyses of p75 cell surface expression upon ZEB1 overexpression, after 10 days treatment with or without 150 nM PLX4032. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's Exact Test).


( G ) Control or ZEB1 - overexpressing A375 cells were transfected with control or p75 - siRNA . p75 and MITF expression levels were was analyzed by quantitative PCR after 48 h . mRNA expression levels are represented relatively to control cells transfected with siRNA Control , in which the levels were fixed at 1 ( mean ± SD , n = 2 ) .

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(G) Control or ZEB1-overexpressing A375 cells were transfected with control or p75-siRNA. p75 and MITF expression levels were was analyzed by quantitative PCR after 48 h. mRNA expression levels are represented relatively to control cells transfected with siRNA Control, in which the levels were fixed at 1 (mean ± SD, n=2).


( H ) Clonogenic assay + / - PLX4032 ( 150 nM ) , + / - GDC - 0973 ( 5 nM ) treatment for 10 days . The graphs represent the mean number of colonies ( ± SD ) in 3 independent experiments ( Student ' s t - test ) . ( I ) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1 - expressing cells ( n = 3 , Student ' s t - test ) .

O O O O O O O O B-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 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

(H) Clonogenic assay +/- PLX4032 (150 nM), +/- GDC-0973 (5 nM) treatment for 10 days. The graphs represent the mean number of colonies (± SD) in 3 independent experiments (Student's t-test). (I) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or ZEB1-expressing cells (n=3, Student's t-test).


( A ) C - 09 . 10 short - term culture cells were infected with retroviruses expressing ZEB1 . Western blot analyses of ZEB1 and p75 . GAPDH was used as a loading control .

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(A) C-09.10 short-term culture cells were infected with retroviruses expressing ZEB1. Western blot analyses of ZEB1 and p75. GAPDH was used as a loading control.