Figure 2A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A 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).


Figure 3A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A 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).


Figure 4A 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 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A 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).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).


Figure 5A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A 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).


Figure 6A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A 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)


Figure 1B ) 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 .

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Figure 1B) 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.


Figure 2B ) UMAP feature plots displaying the normalized expression of CD4 , CD8A and CD8B genes . Data information : Each dot represents one cell .

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Figure 2B) UMAP feature plots displaying the normalized expression of CD4, CD8A and CD8B genes. Data information: Each dot represents one cell.


Figure 3C ) 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 MyositisE ) 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 MyositisF ) Isotype mouse and rabbit IgG1 immunofluorescence stainings . IBM : Inclusion Body MyositisG ) 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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Figure 3C) 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 MyositisE) 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 MyositisF) Isotype mouse and rabbit IgG1 immunofluorescence stainings. IBM: Inclusion Body MyositisG) 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


Figure 4A ) 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 .

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Figure 4A) 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.


Figure 6B ) 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 ;

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Figure 6B) 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;


Figure 1 ( 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 . ( 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 . ( 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 .

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Figure 1(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.(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.(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.


Figure 2 ( 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 . ( 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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Figure 2(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.(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.


Figure 3 ( 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 . ( 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 .

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Figure 3(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.(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.


Figure 4 ( 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 ) . ( 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 ) . ( 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 ) . ( 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 ) .

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Figure 4(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).(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).(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).(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).


Figure 5 ( 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 . ( B ) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression . mRNA expression levels are represented relatively to control cells ( mean ± SD , n = 3 , Student ' s t - test ) . ( C ) Soft agar colony formation assay following ZEB1 expression . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - test ) . ( D ) FACS analyses of p75 cell surface expression upon ZEB1 overexpression . Bar chart representing the mean percentage of p75 - high , int , low and negative cells from 2 independent experiments ( Fisher ' s exact test ) . ( E ) Clonogenic assay + / - PLX4032 ( 100 nM ) treatment for 10 days . The graph represents the mean number of colonies in 3 independent experiments ( Student ' s t - test ) .

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Figure 5(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.(B) Quantitative PCR analyses of MITF and p75 upon ZEB1 expression. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).(C) Soft agar colony formation assay following ZEB1 expression. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).(D) FACS analyses of p75 cell surface expression upon ZEB1 overexpression. Bar chart representing the mean percentage of p75-high, int, low and negative cells from 2 independent experiments (Fisher's exact test).(E) Clonogenic assay +/- PLX4032 (100 nM) treatment for 10 days. The graph represents the mean number of colonies in 3 independent experiments (Student's t-test).


Figure 6 ( A ) A375 cells were infected with retroviruses expressing a control or ZEB1 - shRNA . Western blot analyses of ZEB1 and p75 upon ZEB1 knock - down . GAPDH was used as a loading control . High and low exposures ( exp ) for p75 are shown . ( B ) Quantitative PCR analyses of MITF , p75 , ABCB5 and JARID1B upon ZEB1 knock - down . mRNA expression levels are represented relatively to shRNA control cells ( mean ± SD , n = 3 , Student ' s t - test ) . ( C ) FACS analyses of p75 expression upon ZEB1 knock - down . Bar chart representing the mean percentage of p75 - high , int and low cells from 2 independent experiments ( Fisher ' s exact test ) . ( D ) Soft agar colony formation assay upon ZEB1 knock - down . Scale bar = 200 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - test ) . ( E ) 2 x 106 Control or ZEB1 - shRNA A375 cells were injected subcutaneously into nude mice . The mean tumor volume for 5 mice is represented ( ± SEM ) . ( Student ' s t - test ) . ( F ) A375 cells were infected with an IPTG - inducible ZEB1 - shRNA . Left panel : Western blot analyses of ZEB1 expression ± IPTG ( 100µM ) treatment for 6 days . Actin was used as a loading control . Right panel : 2 x 106 IPTG - inducible shRNA - Control or shRNA - ZEB1A375 cells were injected subcutaneously into nude mice . When the tumor reached 5 mm in diameter , ZEB1 expression was silenced by adding IPTG ( 10 mM ) into the drinking water for 20 days . The mean tumor volume for 5 mice is represented ( ± SEM ) . ( Student ' s t - test ) . ( G ) SKMEL5 cells expressing an IPTG - inducible ZEB1 - shRNA were treated with IPTG ( 100µM ) for 10 days ( + IPTG ) , then IPTG was removed ( - IPTG ) and ZEB1 , MITF and p75 expression levels were analyzed by Western blot and / or quantitative PCR analyses . GAPDH was used as a protein loading control , mRNA expression levels are represented relatively to untreated cells , in which the levels were fixed at 1 ( mean ± SD , n = 2 ) .

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Figure 6(A) A375 cells were infected with retroviruses expressing a control or ZEB1-shRNA. Western blot analyses of ZEB1 and p75 upon ZEB1 knock-down. GAPDH was used as a loading control. High and low exposures (exp) for p75 are shown.(B) Quantitative PCR analyses of MITF, p75, ABCB5 and JARID1B upon ZEB1 knock-down. mRNA expression levels are represented relatively to shRNA control cells (mean ± SD, n=3, Student's t-test).(C) FACS analyses of p75 expression upon ZEB1 knock-down. Bar chart representing the mean percentage of p75-high, int and low cells from 2 independent experiments (Fisher's exact test).(D) Soft agar colony formation assay upon ZEB1 knock-down. Scale bar = 200 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test).(E) 2 x 106 Control or ZEB1-shRNA A375 cells were injected subcutaneously into nude mice. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).(F) A375 cells were infected with an IPTG-inducible ZEB1-shRNA. Left panel: Western blot analyses of ZEB1 expression ± IPTG (100µM) treatment for 6 days. Actin was used as a loading control. Right panel: 2 x 106 IPTG-inducible shRNA-Control or shRNA-ZEB1A375 cells were injected subcutaneously into nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by adding IPTG (10 mM) into the drinking water for 20 days. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).(G) SKMEL5 cells expressing an IPTG-inducible ZEB1-shRNA were treated with IPTG (100µM) for 10 days (+IPTG), then IPTG was removed (-IPTG) and ZEB1, MITF and p75 expression levels were analyzed by Western blot and/or quantitative PCR analyses. GAPDH was used as a protein loading control, mRNA expression levels are represented relatively to untreated cells, in which the levels were fixed at 1 (mean ± SD, n=2).


Figure 7 ( A ) Western blot analyses of ZEB1 and P - ERK in shRNA - control or shRNA - ZEB1 - expressing A375 cells + / - 150 nM PLX4032 treatment for 24 h . GAPDH was used as a loading control . ( B ) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 ( 150 nM ) . Scale bar = 100 µm . Histograms represent quantitative analyses ( mean ± SD , n = 3 , Student ' s t - test ) . ( C ) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA - ZEB1 - expressing cells ( n = 3 , Student ' s t - test ) . ( D ) RPMI7951 , A375 - R and SKMEL5 - R were infected with a retrovirus encoding a constitutive shRNA - ZEB1 . Short - term cultures of GOKA and ESP cells , derived from vemurafenib - resistant patients , were infected with a lentivirus encoding an IPTG - inducible shRNA - ZEB1 . Western blot analyses showing efficient ZEB1 knock - down in the different models , ± IPTG ( 200µM ) , ± PLX4032 ( 3µM ) as indicated . Induction of cell death was assessed by PARP cleavage . GAPDH or actin were used as loading control . ( E ) Clonogenic assays in the presence of 3 µM PLX4032 , and with or without IPTG ( 200 µM ) as indicated . The graphs represent the mean number of colonies in 3 independent experiments ( Student ' s t - test ) . ( F ) Western blot analyses of ZEB1 , p75 and quantitative PCR analyses of MITF in shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant patient - derived short - term culture cells . Actin was used as a loading control . mRNA expression levels are represented relatively to control cells ( mean ± SD , n = 3 , Student ' s t - test ) . ( G ) 2 , 5 x 106 shRNA - Control or shRNA - ZEB1ESPvemurafenib - resistant cells were injected subcutaneously in nude mice . When the tumor reached 5 mm in diameter , ZEB1 expression was silenced by providing mice with IPTG ( 10 mM ) in their drinking water and orally administering vemurafenib ( 50 mg / kg ) daily for 4 weeks . The mean tumor volume for 5 mice is represented ( ± SEM ) . ( Student ' s t - test ) . ( H ) Upper part : Western blot analyses of ZEB1 in shRNA - Control ( 1 to 4 ) or shRNA - ZEB1 ( 5 to 8 ) ESPxenografttumors , showing efficient ZEB1 knock - down directly in the tumors , after IPTG ± PLX4032 treatment in vivo . Lower part : Representative pictures of ZEB1 immunostaining in shRNA - Control or shRNA - ZEB1tumors , after IPTG treatment in vivo . Scale bar = 40µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7(A) Western blot analyses of ZEB1 and P-ERK in shRNA-control or shRNA-ZEB1-expressing A375 cells +/- 150 nM PLX4032 treatment for 24 h. GAPDH was used as a loading control.(B) Soft agar colony formation assay in A375 cells in the presence or absence of PLX4032 (150 nM). Scale bar = 100 µm. Histograms represent quantitative analyses (mean ± SD, n=3, Student's t-test). (C) Number of weeks of chronic exposure to PLX4032 before emergence of resistance in control or shRNA-ZEB1-expressing cells (n=3, Student's t-test).(D) RPMI7951, A375-R and SKMEL5-R were infected with a retrovirus encoding a constitutive shRNA-ZEB1. Short-term cultures of GOKA and ESP cells, derived from vemurafenib-resistant patients, were infected with a lentivirus encoding an IPTG-inducible shRNA-ZEB1. Western blot analyses showing efficient ZEB1 knock-down in the different models, ±IPTG (200µM), ±PLX4032 (3µM) as indicated. Induction of cell death was assessed by PARP cleavage. GAPDH or actin were used as loading control.(E) Clonogenic assays in the presence of 3 µM PLX4032, and with or without IPTG (200 µM) as indicated. The graphs represent the mean number of colonies in 3 independent experiments (Student's t-test).(F) Western blot analyses of ZEB1, p75 and quantitative PCR analyses of MITF in shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant patient-derived short-term culture cells. Actin was used as a loading control. mRNA expression levels are represented relatively to control cells (mean ± SD, n=3, Student's t-test).(G) 2,5 x 106 shRNA-Control or shRNA-ZEB1ESPvemurafenib-resistant cells were injected subcutaneously in nude mice. When the tumor reached 5 mm in diameter, ZEB1 expression was silenced by providing mice with IPTG (10 mM) in their drinking water and orally administering vemurafenib (50 mg/kg) daily for 4 weeks. The mean tumor volume for 5 mice is represented (± SEM). (Student's t-test).(H) Upper part: Western blot analyses of ZEB1 in shRNA-Control (1 to 4) or shRNA-ZEB1 (5 to 8) ESPxenografttumors, showing efficient ZEB1 knock-down directly in the tumors, after IPTG ±PLX4032 treatment in vivo. Lower part: Representative pictures of ZEB1 immunostaining in shRNA-Control or shRNA-ZEB1tumors, after IPTG treatment in vivo. Scale bar = 40µm.


Figure 2 ( A - C ) Telophase GSCs marked by H3S10p ( gray ) expressing old ( green ) and new ( red ) histone H3 show non - overlapping patterns ( A ) . Telophase GSCs expressing old ( green ) and new ( red ) histone H4 show moderate levels of overlap ( B ) . GSCs expressing old ( green ) and new ( red ) histone H2A show high levels of overlap ( C ) . ( D ) A control where the old and new fluorescent tags have been switched shows the non - overlapping patterns of old ( red ) versus new ( green ) H3Rev histone in mitotic telophase GSCs marked by H3S10p ( gray ) . ( E ) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division , where a value of 0 is equal inheritance at exactly a 1 : 1 ratio . Data gathered over 9 separate experiments for H3Rev GSCs , H2A GSCs , and H4 GSCs . Data gathered over 16 separate experiments for H3 GSCs . Values are mean + 95 % CI . ( F - H ) Telophase CBs marked by H3S10p ( gray ) expressing old ( green ) and new ( red ) histone H3 also show non - overlapping patterns . ( F ) Telophase CBs expressing old ( green ) and new ( red ) histone H4 ( G ) and H2A ( H ) , which depict more overlap like GSCs . ( I ) A control with switched old and new fluorescent tags shows the non - overlapping old old ( red ) versus new ( green ) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p ( gray ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(A-C) Telophase GSCs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 show non-overlapping patterns (A). Telophase GSCs expressing old (green) and new (red) histone H4 show moderate levels of overlap (B). GSCs expressing old (green) and new (red) histone H2A show high levels of overlap (C). (D) A control where the old and new fluorescent tags have been switched shows the non-overlapping patterns of old (red) versus new (green) H3Rev histone in mitotic telophase GSCs marked by H3S10p (gray). (E) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev GSCs, H2A GSCs, and H4 GSCs. Data gathered over 16 separate experiments for H3 GSCs. Values are mean + 95% CI. (F-H) Telophase CBs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 also show non-overlapping patterns. (F) Telophase CBs expressing old (green) and new (red) histone H4 (G) and H2A (H), which depict more overlap like GSCs. (I) A control with switched old and new fluorescent tags shows the non-overlapping old old (red) versus new (green) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p (gray).


Figure 3 ( B - C ) Non - overlapping patterns of old ( green ) versus new ( red ) histone H3 ( B ) and overlapping patterns for H2A ( C ) in mitotic prometaphase bam mutant cells marked by anti - H3S10p ( gray ) , similar to WT GSCs . Scale bars : 2μm . ( F - H ) Examples showing old ( green ) versus new ( red ) H3 distribution patterns and percentage of signal strength at a single genomic locus labeled with fluorescent probes ( gray ) for dad gene ( F ) , bgcn gene ( G ) , and ss gene ( H ) in bam mutant GSC - like cells at prometaphase marked by anti - H3S10p ( blue ) . Scale bars : 1μm . Zoomed - in images of each probe are displayed to the right of each figure panel . Scale bars : 0 . 1μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(B-C) Non-overlapping patterns of old (green) versus new (red) histone H3 (B) and overlapping patterns for H2A (C) in mitotic prometaphase bam mutant cells marked by anti-H3S10p (gray), similar to WT GSCs. Scale bars: 2μm.(F-H) Examples showing old (green) versus new (red) H3 distribution patterns and percentage of signal strength at a single genomic locus labeled with fluorescent probes (gray) for dad gene (F), bgcn gene (G), and ss gene (H) in bam mutant GSC-like cells at prometaphase marked by anti-H3S10p (blue). Scale bars: 1μm. Zoomed-in images of each probe are displayed to the right of each figure panel. Scale bars: 0.1μm.


Figure 4 ( A - B ) Old ( red ) versus new ( green ) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes ( cyan ) for dad ( A ) and bam ( B ) in WT telophase female GSCs , marked by anti - H3S10p ( gray ) . ( C ) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs . Data gathered over 8 separate experiments . D

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A-B) Old (red) versus new (green) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes (cyan) for dad (A) and bam (B) in WT telophase female GSCs, marked by anti-H3S10p (gray). (C) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs. Data gathered over 8 separate experiments. D


Figure 1A . Labeling of non - permeabilized ( top ) and permeabilized ( bottom ) cells demonstrate that surface labeling does not label internal proteins . GluA2 labeling ( green ) is visible both on the surface and internally , while DARPP32 labeling ( red ) is only visible internally . B . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no treatment or 20 min 2APB and dantrolene . Bottom . Quantified expression of GluA2 under both conditions , n = 9 replicates of 10 cells each , Student ' s t test . C . Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co - cultures with either no treatment , 30 min DHPG , or 2APB and dantrolene pretreatment followed by DHPG . D . Quantified expression of GluA1 under each condition , n = 9 replicates of 10 cells , p < 0 . 001 , ANOVA . E . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures under the same conditions as in C . F . Quantified expression of GluA2 under each condition , n = 12 replicates of 10 cells each , p < 0 . 001 , ANOVA . G . Representative images of surface myc - GluA2 , internal myc - GluA2 , and DARPP32 expression in striatal neurons from co - cultures under the same conditions as in C . H . Quantified expression of surface myc : total myc under each condition , n = 4 replicates of 10 cells each , p < 0 . 05 , ANOVA . All error bars represent s . e . m . Abbr . A1 = GluA1 , A2 = GluA2 , D = DARPP32 , s - myc = surface myc , i - myc = internal myc , m = merge .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A. Labeling of non-permeabilized (top) and permeabilized (bottom) cells demonstrate that surface labeling does not label internal proteins. GluA2 labeling (green) is visible both on the surface and internally, while DARPP32 labeling (red) is only visible internally.B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student's t test.C. Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co-cultures with either no treatment, 30 min DHPG, or 2APB and dantrolene pretreatment followed by DHPG. D. Quantified expression of GluA1 under each condition, n = 9 replicates of 10 cells, p < 0.001, ANOVA. E. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. F. Quantified expression of GluA2 under each condition, n = 12 replicates of 10 cells each, p < 0.001, ANOVA. G. Representative images of surface myc-GluA2, internal myc-GluA2, and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. H. Quantified expression of surface myc:total myc under each condition, n = 4 replicates of 10 cells each, p < 0.05, ANOVA. All error bars represent s.e.m. Abbr. A1 = GluA1, A2 = GluA2, D = DARPP32, s-myc = surface myc, i-myc = internal myc, m = merge.


Figure 2A . Left . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) or for 30 min with DHPG and pretreated with Xestospungin C and S107 ( XeC / S107 + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 or 20 cells each , p < 0 . 0001 , Student ' s t test . B . Left . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) , or for 30 min with DHPG and pretreated with S107 ( S107 + DHPG ) , or for 30 min with DHPG and pretreated with Xestospungin C ( XeC + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 cells each , ANOVA . C . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures under control or 10 min Thapsigargin ( TG ) treatment . Bottom . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 cells each , Student ' s t test . D . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with treatment with either no DHPG or with DHPG for 30 min , after 15 min of Thapsigargin . Bottom . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 cells each , Student ' s t test . E . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min , after 5 min of BAPTA . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 cells each , p < 0 . 001 , Student ' s t test . F . Top . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA - AM . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 cells each , Student ' s t test . All error bars represent s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student's t test.B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA.C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student's t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student's t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


Figure 3A . Top . Representative image of a radiograph of mature and immature bands of GluA2 after no treatment , DHPG , or 2APB / dan followed by DHPG and incubated with Endo H . Bottom . Quantified graph of the ratio of the mature , endo H insensitive GluA2 band to the total GluA2 for each condition . For DHPG , n = 16 ; for DHPG + 2APB / dan , n = 11 , p < 0 . 05 , ANOVA . B . Top . Representative image of a radiograph of mature and immature bands of GluA1 after no treatment , DHPG , or 2APB / dan followed by DHPG and incubated with Endo H . Bottom . Quantified graph of the ratio of the mature , endo H insensitive GluA1 band to the total GluA1 for each condition , For DHPG , n = 4 ; for DHPG + 2APB / dan , n = 4 , ANOVA . C . Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co - cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment . D . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 cells each , Student ' s t test . All error bars represent s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A. Top. Representative image of a radiograph of mature and immature bands of GluA2 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA2 band to the total GluA2 for each condition. For DHPG, n = 16; for DHPG + 2APB/dan, n = 11, p < 0.05, ANOVA.B. Top. Representative image of a radiograph of mature and immature bands of GluA1 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA1 band to the total GluA1 for each condition, For DHPG, n = 4; for DHPG + 2APB/dan, n = 4, ANOVA.C. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment. D. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


Figure 4A Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment , 10 min DHPG , and 2APB / dantrolene followed by 10 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions . For 10 min DHPG , n = 8 ; for 10 min DHPG + 2APB / dan , n = 6 , p < 0 . 05 , ANOVA . B . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment , 10 min DHPG , and and cycloheximide followed by 10 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions , For 10 min DHPG , n = 8 ; for 10 min DHPG + CHX , n = 5 , p < 0 . 05 , ANOVA . C . Top . Representative immunoblots from GluA2 co - IPs probed for Sec23 and GluA2 after no treatment , 30 min DHPG , and 2APB / dantrolene followed by 30 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 17 ; For 30 min DHPG + 2APB / dan , n = 14 , p < 0 . 05 , ANOVA . D . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment , 30 min DHPG , and cycloheximide followed by 30 min DHPG . Bottom . Quantification of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 12 ; For 30 min DHPG + CHX , n = 11 p < 0 . 01 , ANOVA . E . Top . Representative immunoblot demonstrating IgG and GluA2 coIP after no treatment and 30 min DHPG . Bottom . Quantification of Sec23 after subtracting the non - specific signal , n = 3 , p < 0 . 01 , Student ' s t test . F . Representative immunoblot of a control coIP that demonstrates the specificity of the GluA2 coIP . All error bars represent s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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 4A Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and 2APB/dantrolene followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 10 min DHPG, n = 8; for 10 min DHPG + 2APB/dan, n = 6, p < 0.05, ANOVA.B. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and and cycloheximide followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions, For 10 min DHPG, n = 8; for 10 min DHPG + CHX, n = 5, p < 0.05, ANOVA.C. Top. Representative immunoblots from GluA2 co-IPs probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and 2APB/dantrolene followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 17; For 30 min DHPG + 2APB/dan, n = 14, p < 0.05, ANOVA.D. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and cycloheximide followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 12; For 30 min DHPG + CHX, n = 11 p < 0.01, ANOVA.E. Top. Representative immunoblot demonstrating IgG and GluA2 coIP after no treatment and 30 min DHPG. Bottom. Quantification of Sec23 after subtracting the non-specific signal, n = 3, p < 0.01, Student's t test.F. Representative immunoblot of a control coIP that demonstrates the specificity of the GluA2 coIP. All error bars represent s.e.m.


Figure 5A . Representative immunoblots from GluA2 coIP probed for Sec23 , Sec13 , and GluA2 after no DHPG , 10 , 30 , and 60 minutes of treatment with DHPG . B . Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A . For Sec23 , 10 min DHPG , n = 16 ; 30 min DHPG , n = 15 ; 60 min DHPG , n = 7 , p < 0 . 0001 , ANOVA . For Sec13 , 10 min DHPG , n = 7 ; 30 min DHPG , n = 14 ; 60 min DHPG , n = 6 , p < 0 . 01 , ANOVA . C . Representative images of total expression levels of Sec23 ( top ) and Sec13 ( bottom ) . Expression levels do not change in the presence of DHPG . All error bars represent s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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 5A. Representative immunoblots from GluA2 coIP probed for Sec23, Sec13, and GluA2 after no DHPG, 10, 30, and 60 minutes of treatment with DHPG. B. Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A. For Sec23, 10 min DHPG, n = 16; 30 min DHPG, n = 15; 60 min DHPG, n = 7, p < 0.0001, ANOVA. For Sec13, 10 min DHPG, n = 7; 30 min DHPG, n = 14; 60 min DHPG, n = 6, p < 0.01, ANOVA.C. Representative images of total expression levels of Sec23 (top) and Sec13 (bottom). Expression levels do not change in the presence of DHPG. All error bars represent s.e.m.


Figure 6A . Individual traces of mEPSCs of both control and naspm treated neurons after TTX , TTX + DHPG , and TTX + DHPG + 2APB / dan treatment . B . Averaged mEPSCs and an enlarged time scale from cells under the same conditions as A . Traces from cells that were not treated with naspm are in black and naspm treated traces are in grey . C . Average amplitude of mEPSCs ( pA ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 17 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 005 , ANOVA . D . Average frequency of mEPSC ( Hz ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 12 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 0001 , ANOVA . E . Average amplitude of mEPSCs ( pA ) for only naspm treated neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . F . Average frequency of mEPSC ( Hz ) for only naspm treated neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . G . Average rise time of mEPSC ( ms ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . H . Average decay time of mEPSC ( ms ) for both control ( white bars ) and naspm treated ( black bars ) neurons under each of the conditions , TTX , TTX + DHPG , and TTX + DHPG + 2APB / dantrolene . All error bars represent s . e . m .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A. Individual traces of mEPSCs of both control and naspm treated neurons after TTX, TTX + DHPG, and TTX + DHPG + 2APB/dan treatment.B. Averaged mEPSCs and an enlarged time scale from cells under the same conditions as A. Traces from cells that were not treated with naspm are in black and naspm treated traces are in grey.C. Average amplitude of mEPSCs (pA) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 17; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.005, ANOVA. D. Average frequency of mEPSC (Hz) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 12; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.0001, ANOVA.E. Average amplitude of mEPSCs (pA) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. F. Average frequency of mEPSC (Hz) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene.G. Average rise time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. H. Average decay time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. All error bars represent s.e.m.


Figure 1 . ( a , b ) Dual positive cells are more prevalent in epithelial organs and cells . ( a ) Proportion of ACE2 + TMPRSS2 + cells ( y axis ) per dataset ( dots ) from 21 tissues and organs ( rows ) . ( e - i ) In situ validation of dual positive cells in the lung , airways , and submucosal gland . ( e - g ) PLISH and immunostaining in human adult lung alveoli for ( e ) ACE2 ( white ) , TMPRSS2 ( green ) and CTSL ( red ) , ( f ) ACE2 ( white ) , TMPRSS2 ( green ) and HTII - 280 ( red ) , ( g ) ACE2 ( red ) and Pro - SFTPC ( green ) . ( h ) PLISH and immunostaining in human large airway submucosal glands . ACE2 ( red ) , ACTA2 ( green ) and DAPI ( blue ) . ( i ) PLISH in human adult large airway . ACE2 ( green ) and DAPI ( blue ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1.(a,b) Dual positive cells are more prevalent in epithelial organs and cells. (a) Proportion of ACE2+TMPRSS2+ cells (y axis) per dataset (dots) from 21 tissues and organs (rows).(e-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).


Figure 2 . ( a , c ) Single - cell ATAC - seq of lung samples from primary carina ( 1C ) and subpleural parenchyme ( RPL ) ( n = 1 patient , k = 3 samples , 3 , 366 cells from 1C , 8 , 340 cells from RPL ) . Uniform Manifold Approximation and Projection ( UMAP ) embedding of scATAC - seq profiles ( dots ) colored by ( a , left ) cell types , ( a , right ) cells with at least 1 fragment ( indicating accessibility , open chromatin ) mapping to the ACE2 gene locus ( defined as − 2kb upstream the Transcription Start Site to Transcription End Site ) , or by sample

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Figure 2.(a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location


Figure 3 . ( g ) Low expression in pediatric samples . Mean expression level ( log CPM , y axis ) of ACE2 ( blue ) , TMPRSS2 ( orange ) , and CTSL ( green ) across age bins ( x axis ) in AT2 ( left ) and ciliated ( right ) cells . Pediatric samples : 0 - 10 years . Samples from past or current smokers were removed from this plot to avoid smoking confounders . Error bars are omitted due to y - axis limitations . They are typically 10 - fold the mean value ( Supplementary Table 5 ) . Multiciliated and AT2 cells are shown as these cell types are present in fetal data , and show significant age associations with ACE2 expression .

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Figure 3.(g) Low expression in pediatric samples. Mean expression level (log CPM, y axis) of ACE2 (blue), TMPRSS2 (orange), and CTSL (green) across age bins (x axis) in AT2 (left) and ciliated (right) cells. Pediatric samples: 0-10 years. Samples from past or current smokers were removed from this plot to avoid smoking confounders. Error bars are omitted due to y-axis limitations. They are typically 10-fold the mean value (Supplementary Table 5). Multiciliated and AT2 cells are shown as these cell types are present in fetal data, and show significant age associations with ACE2 expression.


Figure 5 : ( a ) Gradual increase in Ace2 expression by airway epithelial cell type with age . Mean expression ( y axis ) of Ace2 in different airway epithelial cells ( x axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , and error bars ( standard error of the mean ( SEM ) ) . ( b ) Increase in proportion of Ace2 + Ctsl + goblet and club cells with age . Percent of Ace2 + Ctsl + cells ( x axis ) in different airway epithelial cell types ( y axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , and error bars ( SEM ) . ( c - j ) Increase in Ace2 expression in secretory cells with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( c , d ) UMAP of scRNA - seq profiles ( dots ) colored by experimental group ( c ) or by Ace2 + cells and indicated double positive cells ( d ) . Alveolar epithelial cells ( AT1 and AT2 ) and airway epithelial secretory and ciliated cells are marked . ( c - j ) Increase in Ace2 expression in secretory cells with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( e ) The relative frequency of Ace2 + cells is increased by smoking in airway secretory cells but not AT2 cells . Relative proportion ( y axis ) of Ace2 + ( red ) and Ace2 - ( grey ) cells in smoking and control mice of different cell types ( x axis ) . ( f , g ) Expression of Ace2 is increased in airway secretory cells , but not in AT2 cells . Distribution of Ace2 expression ( y axis ) in secretory ( f ) and AT2 ( g ) cells from control and smoking mice ( x axis ) . ( h - j ) Re - analysis of published bulk mRNA - Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of ( h ) Ace2 , ( i ) Tmprss2 , and ( j ) Ctsl after five months of chronic exposure . ( k ) Expression in placenta . UMAP plots showing expression of Ace2 , Tmprss2 and Ctsl in single and double positive cells from embryonic days 9 . 5 to 18 ( left ) and embryonic day 14 . 5 ( right ) of mouse placenta

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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:(a) Gradual increase in Ace2 expression by airway epithelial cell type with age. Mean expression (y axis) of Ace2 in different airway epithelial cells (x axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (standard error of the mean (SEM)).(b) Increase in proportion of Ace2+Ctsl+ goblet and club cells with age. Percent of Ace2+Ctsl+ cells (x axis) in different airway epithelial cell types (y axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (SEM).(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (c,d) UMAP of scRNA-seq profiles (dots) colored by experimental group (c) or by Ace2+ cells and indicated double positive cells (d). Alveolar epithelial cells (AT1 and AT2) and airway epithelial secretory and ciliated cells are marked.(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (e) The relative frequency of Ace2+ cells is increased by smoking in airway secretory cells but not AT2 cells. Relative proportion (y axis) of Ace2+ (red) and Ace2- (grey) cells in smoking and control mice of different cell types (x axis).(f, g) Expression of Ace2 is increased in airway secretory cells, but not in AT2 cells. Distribution of Ace2 expression (y axis) in secretory (f) and AT2 (g) cells from control and smoking mice (x axis).(h-j) Re-analysis of published bulk mRNA-Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of (h) Ace2, (i) Tmprss2, and (j) Ctsl after five months of chronic exposure.(k) Expression in placenta. UMAP plots showing expression of Ace2, Tmprss2 and Ctsl in single and double positive cells from embryonic days 9.5 to 18 (left) and embryonic day 14.5 (right) of mouse placenta development


Figure 6 . ( a ) Multiple proteases are co - expressed with ACE2 in human lung scRNA - seq . Scatter plot of significance ( y axis , - log10 ( adjusted P value ) ) and effect size ( x axis ) of co - expression of each protease gene ( dot ) with ACE within each indicated epithelial cell type ( color ) . Dashed line : significance threshold . TMPRSS2 and PCSKs that significantly co - expressed with ACE2 are marked .

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Figure 6.(a) Multiple proteases are co-expressed with ACE2 in human lung scRNA-seq. Scatter plot of significance (y axis, -log10(adjusted P value)) and effect size (x axis) of co-expression of each protease gene (dot) with ACE within each indicated epithelial cell type (color). Dashed line: significance threshold. TMPRSS2 and PCSKs that significantly co-expressed with ACE2 are marked.