Figure 4A Lines based on the human melanoma cell line 1205Lu ( BRAF - V600E - positive ) were made to carry GFP - Usp27x , GFP - Usp27xC87A or GFP - Usp22 under the control of a doxycycline ( dox ) - inducible promoter . GFP - Usp27x , GFP - Usp27xC87A or GFP - Usp22 were induced with dox for 24 or 48h . Levels of Bim were determined by Western blotting . The two left panels are from the same membrane and exposure times . Similar results were obtained in n = 4 separate experiments ( left ) and n = 3 experiments ( right ) . See also Appendix Fig . S6A where 3xFlag - Usp22 is also shown to be unable to increase Bim levels . B A cell line derived from the BRAF - V600E - positive human melanoma line WM1158 was generated to carry inducible GFP - Usp27x . GFP - Usp27x was induced , and Bim was detected as in A . Similar results were obtained in n = 3 separate experiments . C Derivatives of the NSCLC cell line HCC827 ( expressing constitutively active EGFR ) were generated that only express the TetR - repressor or carrying in addition either GFP - Usp27x or GFP - Usp27xC87A . Cells were treated with dox for 72h . Bim was detected by Western blotting . Similar results were obtained in n = 2 separate experiments . GFP - Usp27x , GFP - Usp27xC87A , or GFP - Usp22 expression ( A - C ) was detected using an antibody against GFP . D Caco2 cells carrying a dox inducible HA - BRAF - V600E [ 37 ] ( left two lanes ) or the same cells stably expressing 3xFlag - Usp27x were treated with dox for 48h to induce expression of HA - BRAF - V600E . Bim was detected by Western blotting . The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100 % ( normalized to the tubulin signal for each condition ) . Similar results were obtained in n = 3 separate experiments with varying induction times . See also Appendix Fig . S6D .

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Figure 4A Lines based on the human melanoma cell line 1205Lu (BRAF-V600E-positive) were made to carry GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 under the control of a doxycycline (dox)-inducible promoter. GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 were induced with dox for 24 or 48h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n=4 separate experiments (left) and n=3 experiments (right). See also Appendix Fig. S6A where 3xFlag-Usp22 is also shown to be unable to increase Bim levels.B A cell line derived from the BRAF-V600E-positive human melanoma line WM1158 was generated to carry inducible GFP-Usp27x. GFP-Usp27x was induced, and Bim was detected as in A. Similar results were obtained in n=3 separate experiments.C Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the TetR-repressor or carrying in addition either GFP-Usp27x or GFP-Usp27xC87A. Cells were treated with dox for 72h. Bim was detected by Western blotting. Similar results were obtained in n=2 separate experiments. GFP-Usp27x, GFP-Usp27xC87A, or GFP-Usp22 expression (A-C) was detected using an antibody against GFP.D Caco2 cells carrying a dox inducible HA-BRAF-V600E [37] (left two lanes) or the same cells stably expressing 3xFlag-Usp27x were treated with dox for 48h to induce expression of HA-BRAF-V600E. Bim was detected by Western blotting. The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n=3 separate experiments with varying induction times. See also Appendix Fig. S6D.


Figure 5A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF - V600ECaco2 cells carrying inducible HA - BRAF - V600E were transfected with control siRNA ( siCo3 ) or siRNA directed against Usp27x ( combination of three siRNAs targeting Usp27xmRNA ) for 24h prior to BRAF - V600E expression with dox for 2h ( left ) or 4h ( right ) . BimEL was detected by Western blotting . Similar results were also observed in n = 2 more experiments after 3h induction ( not shown , see Appendix Fig . S6E for siRNA efficacy ) . B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT - Usp27xKO ( clone 2 / 10 , generated using CRISPR - Cas - 9 ) were treated with PMA [ 16 . 2 nM ] for 16h or 24h to induce BimEL degradation . Western blots show protein levels of n = 3 independently performed experiments ( similar results were obtained in one more experiment , not shown ) . See Appendix Fig . S6G for verification of the Usp27xKO cell line by DNA sequence analysis .

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Figure 5A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF-V600ECaco2 cells carrying inducible HA-BRAF-V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27xmRNA) for 24h prior to BRAF-V600E expression with dox for 2h (left) or 4h (right). BimEL was detected by Western blotting. Similar results were also observed in n=2 more experiments after 3h induction (not shown, see Appendix Fig. S6E for siRNA efficacy).B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT-Usp27xKO (clone 2/10, generated using CRISPR-Cas-9) were treated with PMA [16.2 nM] for 16h or 24h to induce BimEL degradation. Western blots show protein levels of n=3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Appendix Fig. S6G for verification of the Usp27xKO cell line by DNA sequence analysis.


Figure 6A 293FT - TetR - 3xFlagUsp27x cells were treated with PMA [ 16 . 2 nM ] plus QVD [ 10 µM ] and 3xFlag - Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide ( CHX ) . Cells were harvested at the indicated time points , and levels of BimEL were determined by Western blotting . Similar results were obtained in n = 3 separate experiments . Cut membranes shown were from one membrane with same exposure time . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point . B 1205Lu melanoma cells carrying dox - inducible GFP - Usp27x were treated with dox for 24h . Cycloheximide ( CHX , 1µg / ml ) was then added . Cells were harvested at the indicated time points , and levels of BimEL were determined by Western blotting . GFP - Usp27x was detected with anti - GFP - antibodies . Similar results were obtained in n = 3 separate experiments . A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox - inducible GFP - Usp27xC87A mutant . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point set to 100 % . The starting point for the non - Usp27x / C87A inducing cells ( - dox ) is shown in lane 1 for experiment one and two ( - dox , - CHX ) . Cut membranes shown were from one membrane with same exposure time . Same results were obtained for WM1158 melanoma cells ( data not shown ) . C HCC827 cells were induced to express GFP - Usp27x with dox as indicated . 24h later cycloheximide ( CHX , 1µg / ml ) was added for the indicated time . BimEL was detected after the indicated times of treatment by Western blotting . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point . Similar results were obtained in n = 2 separate experiments . Cut membranes shown were from one membrane with same exposure time .

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Figure 6A 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point.B 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).C HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.


Figure 7A 1205Lu melanoma cells carrying dox - inducible GFP , GFP - Usp27x or GFP - Usp27xC87A were treated with doxycycline ( dox ) as indicated . After 48 hUO126 ( 10µM ) was added for another 48 h . Apoptosis was measured by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = 3 ( GFP ) or from n ≥ 6 separate experiments ( GFP - Usp27x and GFP - Usp27xC87A ) . P - values ( t - test ) for statistically significant differences are shown . N . s . , p > 0 . 05 . The addition of QVD inhibited cells from active Caspase - 3 positive staining ( not shown ) . A stain for active Bax is shown in Appendix Fig . S7B . B HCC827 NSCLC cells carrying dox - inducible GFP - Usp27xC87A , GFP - Usp27x , or two separate lines established from these GFP - Usp27x - cells where the Bim - locus had been targeted for deletion using CRISPR / Cas9 ( Bim - KO ) , were treated with combinations of dox and the EGFR - inhibitor gefitinib as indicated . Apoptosis was measured after 72h of treatment by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = ≥ 15 ( maternal GFP - Usp27x line ) or from n ≥ 5 ( for Bim1KO ) or from n ≥ 6 ( for Bim2KO ) , or n = 4 ( for GFP - Usp27xC87A ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . Again , the addition of QVD inhibited cells from active Caspase - 3 positive staining ( not shown ) . The inset shows Bim - knock - out - efficiency ( n = 2 ) .

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Figure 7A 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 hUO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B.B HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).


Figure 8Figure 8 Loss of Usp27xreduces gefitinib - induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x - locus had been targeted for deletion using CRISPR / Cas9 with two different guide RNA against Usp27x ( Usp27xKO - 1 and Usp27xKO - 4 ) , were treated with gefitinib [ 10µM ] for 48h . Apoptosis was measured by staining for active caspase - 3 ( A ) or by staining for activated Bax ( antibody : 6A7 clone ) followed by flow cytometric analysis . Data shown in A , B ( means / SEM ) are from n = 4 separate experiments . P - values ( t - test ) for statistically significant differences are shown .

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Figure 8Figure 8 Loss of Usp27xreduces gefitinib-induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x-locus had been targeted for deletion using CRISPR/Cas9 with two different guide RNA against Usp27x (Usp27xKO-1 and Usp27xKO-4), were treated with gefitinib [10µM] for 48h. Apoptosis was measured by staining for active caspase-3 (A) or by staining for activated Bax (antibody: 6A7 clone) followed by flow cytometric analysis. Data shown in A, B (means/SEM) are from n=4 separate experiments. P-values (t-test) for statistically significant differences are shown.


Figure 1Western blot detection of PAX6 isoforms in human fetal forebrain and retina . The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight . The green arrow indicates the PAX6A & B isoforms . The red arrow indicates the PAX6D isoform . # 1 , # 2 are # 3 are from ~ 8 - week - old human embryos . # 4 is from ~ 12 - week - old human fetus . qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . qPCR quantification of PAX6A & B isoform expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . qPCR of PAX6D isoform expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs . D10 indicates day 10 of differentiation . The green arrow indicates the PAX6A & B isoforms . The red arrow indicates the PAX6D isoform .

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Figure 1Western blot detection of PAX6 isoforms in human fetal forebrain and retina. The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform. #1, #2 are #3 are from ~8-week-old human embryos. #4 is from ~12-week-old human fetus.qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR quantification of PAX6A&B isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR of PAX6D isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group).Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs. D10 indicates day 10 of differentiation. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform.


Figure 2qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation ( n = 3 biological replicates for each group ) . Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation . Immunostaining of PAX6 in Wildtype and PAX6 KO cells . C ' - PAX6 antibody detects PAX6A , B and D . N ' - PAX6 antibody detects PAX6 A and B but not D . Scale bars : 50 µm . qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells . Samples are collected at day 6 , 10 , 14 and 19 after the start of differentiation .

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Figure 2qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation (n=3 biological replicates for each group).Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation.Immunostaining of PAX6 in Wildtype and PAX6 KO cells. C'-PAX6 antibody detects PAX6A, B and D. N'-PAX6 antibody detects PAX6 A and B but not D. Scale bars: 50 µm.qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells. Samples are collected at day 6, 10, 14 and 19 after the start of differentiation.


Figure 3qPCR verification of PAX6D KO efficiency . PAX6A & B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers ( n = 3 biological replicates for each group ) . Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells . qPCR quantification of retinal gene VSX2 in PAX6D KO cells ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene SIX6 in PAX6D KO cells ( n = 3 biological replicates for each group ) . Immunostaining of VSX2 in wildtype and PAX6D KO cells . Scale bars : 100 µm . Samples were from day 30 differentiating retinal cells .

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Figure 3qPCR verification of PAX6D KO efficiency. PAX6A&B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers (n=3 biological replicates for each group).Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells.qPCR quantification of retinal gene VSX2 in PAX6D KO cells (n=3 biological replicates for each group).   qPCR quantification of retinal gene SIX6 in PAX6D KO cells (n=3 biological replicates for each group).   Immunostaining of VSX2 in wildtype and PAX6D KO cells. Scale bars:100 µm. Samples were from day 30 differentiating retinal cells.


Figure 4Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues . Scale bars : 50 µm . Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype , PAX6 KO and PAX6D KO cell lines . Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype , PAX6 KO and PAX6D KO cell lines . Quantification of SOX1 - / SOX2 + ratio in panel C ( n = 3 biological replicates for each sample ) . qPCR quantification of SOX1 in wildtype , PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation ( n = 3 biological replicates for each sample ) .

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Figure 4Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues. Scale bars:50 µm.Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines. Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines.Quantification of SOX1-/SOX2+ ratio in panel C (n=3 biological replicates for each sample).qPCR quantification of SOX1 in wildtype, PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation (n=3 biological replicates for each sample).


Figure 5qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX ( n = 3 biological replicates for each sample ) . Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX . qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells ( n = 3 biological replicates for each group ) . qPCR quantification of retinal marker expression in PAX6D TetOn cells w / o DOX treatment ( n = 3 biological replicates for each group ) . Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry . SOX1 / SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate .

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Figure 5qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX (n=3 biological replicates for each sample).Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX.qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells (n=3 biological replicates for each group).qPCR quantification of retinal marker expression in PAX6D TetOn cells w/o DOX treatment (n=3 biological replicates for each group).Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry. SOX1/SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate.


Figure 6Verification of PAX6D target genes using PAX6D TetOn cells . Two sets of genes ( retinal and non - retinal neural genes ) are directly regulated by PAX6D in opposite directions ( n = 3 biological replicates for each group ) .

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Figure 6Verification of PAX6D target genes using PAX6D TetOn cells. Two sets of genes (retinal and non-retinal neural genes) are directly regulated by PAX6D in opposite directions (n=3 biological replicates for each group).


Figure 7qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation ( n = 3 biological replicates for each group ) . Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat , control shRNA lentivirus , WNT8B shRNA lentivirus . qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 ( n = 3 biological replicates for each group ) .

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 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 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 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 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

Figure 7qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation (n=3 biological replicates for each group).qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation (n=3 biological replicates for each group).qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation (n=3 biological replicates for each group).Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat, control shRNA lentivirus, WNT8B shRNA lentivirus.qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus (n=3 biological replicates for each group).qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 (n=3 biological replicates for each group).


Figure 1a , TEM analysis of P7 wt retinae ( N = 4 eyes ) showing a ( photoreceptor - Multivesicular Body ) PR - MVB in close proximity to ( Muller glia ) MG ( red dashed box ) . PR - photoreceptor cell , MG ( blue dashed lines ) - muller glia cell , MVB ( yellow and yellow arrow ) - multivesicular body , m ( green ) - mitochondria , cc ( pink ) - connecting cilium , RPE ( purple and purple dashed line ) - retina pigment epithelium , RPE melanin - black arrows . Scale bar = 2µm left , 1µm right ; f , Representative Dot Blot of 100K EV pellet ( each dot represents 3 pooled EV isolations , derived from 60 * 106 cells ; N = 8 experiments ) vs cell lysate ( CL ) from P8 Nrl . Gfp + / + photoreceptors . EV markers = LAMP1 , CD8 , CD9 ; Golgi marker = GM130 ; phototransduction markers = Recoverin , Rhodopsin , Rod α - Transducin ; g , RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1 , Rec , Rho , Crx , Cre and Gfp , relative to β - actin , comparing EVs ( N = 8 ) against the appropriate ( Nrl . Gfp + / + or Nrl . Cre + / - ) photoreceptor cell lysate ( N = 3 ) . Gnat1 , Cre and Gfp mRNA were present in all relevant samples ; i , representative flow cytometry analysis of Nrl . Cre + / - & mTmGfloxed co - cultures . Samples were analyzed for expression of myrGFP ( recombination ) and CD73 ( photoreceptor identity ) versus CD73 - ve fraction ( other retinal cells ) . N > 3 independent cultures for each condition with technical triplicate for each sample ; One way ANOVA , non - parametric , Kruskal - Wallis with Dunns ' multiple comparisons

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 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 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 O O O O

Figure 1a, TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2µm left, 1µm right;f, Representative Dot Blot of 100K EV pellet (each dot represents 3 pooled EV isolations, derived from 60*106 cells; N = 8 experiments) vs cell lysate (CL) from P8 Nrl.Gfp+/+ photoreceptors. EV markers = LAMP1, CD8, CD9; Golgi marker = GM130; phototransduction markers = Recoverin, Rhodopsin, Rod α-Transducin;g, RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1, Rec, Rho, Crx, Cre and Gfp, relative to β-actin, comparing EVs (N = 8) against the appropriate (Nrl.Gfp+/+ or Nrl.Cre+/-) photoreceptor cell lysate (N = 3). Gnat1, Cre and Gfp mRNA were present in all relevant samples;i, representative flow cytometry analysis of Nrl.Cre+/- & mTmGfloxed co-cultures. Samples were analyzed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73-ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test


Figure 2g , Quantification of no . of TdTomato + ve host photoreceptor cells seen following transplantation of live ( 224 ± 181 ) versus UV - treated ( dead ) ( 3 ± 4 ) Nrl . Cre + / - P8 photoreceptors ( N = 4 eyes per condition ) . Graph shows mean ± S . D .

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Figure 2g, Quantification of no. of TdTomato+ve host photoreceptor cells seen following transplantation of live (224 ± 181) versus UV-treated (dead) (3 ± 4) Nrl.Cre+/- P8 photoreceptors (N = 4 eyes per condition). Graph shows mean ± S.D.


Figure 3i & j , Representative images of depth colour coding ( ImageJ ) of x , y , z live imaging of Nrl . Gfp + / + photoreceptor cultures showing ( i ) neurites ( blue arrows ) growing along the substrate ( asterisks denote secondary branching of long GFP + neurites ) , and in ( j ) , a free - floating PhNT ( magenta arrow ) . Scale bar = 10 µm ; k & l , Quantification of the localization of Nrl . Gfp + / + processes in the z - axis ( depth ) ( N = 4 independent cultures , n = 1096 cells , nneurites = 440 , nnanotubes = 31 where ( k ) neurites . P values = * * * * p < 0 . 0001 ( bottom vs top ) ; * * p = 0 . 002 ( bottom vs middle ) ; * * p = 0 . 003 ( middle vs top ) and ( l ) PhNTs ; P - values = * * * * p < 0 . 0001 ( bottom vs middle & middle vs top ) . One way ANOVA , non - parametric , Kruskal - Wallis with Dunns ' multiple comparisons test shows means vary significantly .

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 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

Figure 3i & j, Representative images of depth colour coding (ImageJ) of x,y,z live imaging of Nrl.Gfp+/+ photoreceptor cultures showing (i) neurites (blue arrows) growing along the substrate (asterisks denote secondary branching of long GFP+ neurites), and in (j), a free-floating PhNT (magenta arrow). Scale bar = 10 µm;k & l, Quantification of the localization of Nrl.Gfp+/+ processes in the z-axis (depth) (N = 4 independent cultures, n = 1096 cells, nneurites = 440, nnanotubes = 31 where (k) neurites. P values = **** p < 0.0001 (bottom vs top); **p = 0.002 (bottom vs middle); **p = 0.003 (middle vs top) and (l) PhNTs; P-values = **** p < 0.0001 (bottom vs middle & middle vs top) . One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test shows means vary significantly.


Figure 4d , representative time lapse images of cGFP mobility between two PhNT - connected Nrl . Gfp + / + photoreceptors ( dashed ellipse = bleached area ) versus an isolated cell ( solid ellipse = bleached area ) and clustered cells ( dot ellipse = bleached area ) . Images show pre - bleach ( t = - 5 ' ' ) , bleach ( t = 0 ' ' ) , and post - bleach ( t = 35 ' ' and 215 ' ' ) . Unbleached reference cells also indicated ( arrows ) ; Green = cytoplasm ; Scale bar = 5µm .

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Figure 4d, representative time lapse images of cGFP mobility between two PhNT-connected Nrl.Gfp+/+ photoreceptors (dashed ellipse = bleached area) versus an isolated cell (solid ellipse = bleached area) and clustered cells (dot ellipse = bleached area). Images show pre-bleach (t = -5''), bleach (t = 0''), and post-bleach (t = 35'' and 215''). Unbleached reference cells also indicated (arrows); Green = cytoplasm; Scale bar = 5µm.


Figure 5d , Lysosomes can be exchanged , rarely , between PhNT - connected cells . Lysosomes were labelled with SiR - Lyso and their movements were analysed by TrackMate software . Images show deconvolution of 3D reconstructions of lysosomes ( surface ; red ) and cGFP ( volume ; green ) from time - lapse live imaging series of Nrl . Gfp + / + photoreceptor cultures ( green ) ; the position of a transferred lysosome is marked as1 ( t = 0 ) , 2 ( t = 3 ' ) , 3 ( t = 6 ' ) , 4 ( t = 9 ' ) ; N . B . Deconvolution shows a segment - like process extending from cell " A " and a PhNT connecting cells " A " and " B " ; Scale bar = 2 µm ; e , Mitochondria can be exchanged , rarely , between PhNT - connected cells . Mitochondria movements determined by TrackMate software . Images show deconvolution of 3D reconstructions of mitochondria ( surface ; red ) and cGFP ( volume ; green ) from time - lapse live imaging series ; position of transferred mitochondrion is marked as 1 ( t = 0 ) , 2 ( t = 6 ' ) , 3 ( t = 12 ' ) , 4 ( t = 18 ' ) ; Mitochondria labelled with mito - Tracker - Orange ; Scale bar = 5 µm .

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Figure 5d, Lysosomes can be exchanged, rarely, between PhNT-connected cells. Lysosomes were labelled with SiR-Lyso and their movements were analysed by TrackMate software. Images show deconvolution of 3D reconstructions of lysosomes (surface; red) and cGFP (volume; green) from time-lapse live imaging series of Nrl.Gfp+/+ photoreceptor cultures (green); the position of a transferred lysosome is marked as1 (t = 0), 2 (t = 3'), 3 (t = 6'), 4 (t = 9'); N.B. Deconvolution shows a segment-like process extending from cell "A" and a PhNT connecting cells "A" and "B"; Scale bar = 2 µm;e, Mitochondria can be exchanged, rarely, between PhNT-connected cells. Mitochondria movements determined by TrackMate software. Images show deconvolution of 3D reconstructions of mitochondria (surface; red) and cGFP(volume; green) from time-lapse live imaging series; position of transferred mitochondrion is marked as 1 (t = 0), 2 (t = 6'), 3 (t = 12'), 4 (t = 18'); Mitochondria labelled with mito-Tracker-Orange; Scale bar = 5 µm.


Figure 6a , MIP image of wildtype ( wt ) retina transplanted with P8 Nrl . Gfp + / + CD73 + MACS - enriched photoreceptors ; dashed box indicates ROI subjected to 3D deconvolution and volume reconstruction ( right ) and shows PhNT - like connection between donor and host photoreceptor at the level of host inner segment . Additional images show 3D segmentation of z = 0 and z = 38 focal planes , and reveal the PhNT ; green = GFP , blue = nuclei ; Scale bar = 10 µm in MIP and 5 µm volume reconstruction ; b & c , assessment of properties of PhNT - like processes between donor and host photoreceptors ( n = 19 PhNT - like processes ; N = 10 eyes ) showing ( b ) diameter and ( c ) length ; d , MIP image of wt retina transplanted with P8 Nrl . Gfp + / + x myrRFP / + photoreceptors ; showing wt host ONL photoreceptor labeled with cGFP ( green ) but not with myrRFP ( red ) ; Scale bar = 50µm ; e , quantification and statistical analysis of material transfer events following subretinal transplantation of live ( cGFP = 3775 ± 804 , myrRFP = 436 ± 145 ) or UV - treated ( cGFP = 1 . 3 ± 2 . 5 , myrRFP = 4 . 3 ± 5 . 7 ) Nrl . Gfp + / + x myr - Rfp + / + photoreceptors into wt hosts ( N = 8 and N = 4 retinae , respectively ) ; One - way ANOVA , parametric , two tail , Tukey ' s multiple comparisons ; Graph shows mean ± S . D . f - h , Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscopeTM . f , Representative MIP images of Gnat1 - / - eyes transplanted with Nrl . Gfp + / + donor cells and processed in situ for Gfp mRNA ( red ) and Gnat1 mRNA ( magenta ) ; blue = nuclei ; ROIs indicated in ( f ) show GFP + ve donor cell mass ( CM ) and GFP + ve cells within Gnat1 - / - host outer nuclear layer ( ONL ) ; g & h , quantification of Gfp and Gnat1 mRNA ( mean fluorescence intensity per cell ) , respectively ( N = 3 retinae ) ; One - way ANOVA , non - parametric , two tailed analysis showed no significant differences in signal intensity for either Gnat1 or Gfp mRNA between GFP + ve and GFP - ve host photoreceptors ; i , Manipulation of actin signalling alters material transfer in vivo : ( far left ) MIP image showing GFP + ve host photoreceptor ( green ) expressing Rod α - transducin ( red ) after transplantation of retinal cells transduced with lenti - CAG - P2A . Gfp ( transduction control ) into Gnat1 - / - recipient ; Scale bar = 10 µm ; adjacent images show Gnat1 - / - eyes transplanted with P2 retinal cells transduced with ( left ) plenti - CAG - P2A . GFP ( green ) , or ( middle ) plenti - CAG - RhoA - P2A . GFP ( green ) , or ( right ) plenti - CAG - ΔΝRac1 - P2A . GFP ( green ) ; green = GFP , blue = nuclei ; Scale bar = 20 µm ; j , quantification of the number of GFP + cells in Gnat1 - / - host ONL , ( N = 5 retinae per condition ) ; plenti - CAG - P2A . GFP ( 2328 ± 234 ) , plenti - CAG - RhoA - P2A . GFP ( 668 ± 481 ) , plenti - CAG - ΔΝRac1 - P2A . GFP ( 547 ± 306 ) ; One - way ANOVA , non - parametric , Dunn ' s multiple comparisons test . Graph shows mean ± SD .

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 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 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 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

Figure 6a, MIP image of wildtype (wt) retina transplanted with P8 Nrl.Gfp+/+ CD73+ MACS-enriched photoreceptors; dashed box indicates ROI subjected to 3D deconvolution and volume reconstruction (right) and shows PhNT-like connection between donor and host photoreceptor at the level of host inner segment. Additional images show 3D segmentation of z = 0 and z = 38 focal planes, and reveal the PhNT; green = GFP, blue = nuclei; Scale bar = 10 µm in MIP and 5 µm volume reconstruction; b & c, assessment of properties of PhNT-like processes between donor and host photoreceptors (n = 19 PhNT-like processes; N = 10 eyes) showing (b) diameter and (c) length; d, MIP image of wt retina transplanted with P8 Nrl.Gfp+/+ x myrRFP/+ photoreceptors; showing wt host ONL photoreceptor labeled with cGFP (green) but not with myrRFP (red); Scale bar = 50µm; e, quantification and statistical analysis of material transfer events following subretinal transplantation of live (cGFP = 3775 ± 804, myrRFP = 436 ± 145) or UV-treated (cGFP = 1.3 ± 2.5, myrRFP = 4.3 ± 5.7) Nrl.Gfp+/+x myr-Rfp+/+ photoreceptors into wt hosts (N = 8 and N = 4 retinae, respectively); One-way ANOVA, parametric, two tail, Tukey's multiple comparisons; Graph shows mean ± S.D. f-h, Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscopeTM. f, Representative MIP images of Gnat1-/- eyes transplanted with Nrl.Gfp+/+ donor cells and processed in situ for Gfp mRNA (red) and Gnat1 mRNA (magenta); blue = nuclei; ROIs indicated in (f) show GFP+ve donor cell mass (CM) and GFP+ve cells within Gnat1-/- host outer nuclear layer (ONL); g & h, quantification of Gfp and Gnat1 mRNA (mean fluorescence intensity per cell), respectively (N = 3 retinae); One-way ANOVA, non-parametric, two tailed analysis showed no significant differences in signal intensity for either Gnat1 or Gfp mRNA between GFP+ve and GFP-ve host photoreceptors;i, Manipulation of actin signalling alters material transfer in vivo: (far left) MIP image showing GFP+ve host photoreceptor (green) expressing Rod α-transducin (red) after transplantation of retinal cells transduced with lenti-CAG-P2A.Gfp (transduction control) into Gnat1-/- recipient; Scale bar = 10 µm; adjacent images show Gnat1-/- eyes transplanted with P2 retinal cells transduced with (left) plenti-CAG-P2A.GFP (green), or (middle) plenti-CAG-RhoA-P2A.GFP (green), or (right) plenti-CAG-ΔΝRac1-P2A.GFP (green); green = GFP, blue = nuclei; Scale bar = 20 µm; j, quantification of the number of GFP+ cells in Gnat1-/- host ONL, (N = 5 retinae per condition); plenti-CAG-P2A.GFP (2328 ± 234), plenti-CAG-RhoA-P2A.GFP (668 ± 481), plenti-CAG-ΔΝRac1-P2A.GFP (547 ± 306); One-way ANOVA, non-parametric, Dunn's multiple comparisons test. Graph shows mean ± SD.


Figure 7b , Representative tile scan of un - injected Nrl . Cre + / - / TdTomato + / - chimera showing endogenous recombination ( white ) ; ROIs , as indicated with dashed or dot boxes ; Scale bars = 50 µm ; c & d , ROI from un - injected Nrl . Cre + / - / TdTomato + / - chimera showing spontaneous recombination presenting in mosaic stripe patterning ; Scale bar = 50 µm in c and 10 µm in d ; e & f , Representative images of Nrl . Cre + / - / TdTomato + / - chimera after receiving a subretinal injection of AAV - Nrl . Cre . The injected eyes show widespread recombination , which also shows the anticipated mosaic stripe patterning ; Scale bar = 50 µm in ( e ) and 10 µm in ( f ) ;

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Figure 7b, Representative tile scan of un-injected Nrl.Cre +/-/TdTomato+/- chimera showing endogenous recombination (white); ROIs, as indicated with dashed or dot boxes; Scale bars = 50 µm;c & d, ROI from un-injected Nrl.Cre +/-/TdTomato+/- chimera showing spontaneous recombination presenting in mosaic stripe patterning; Scale bar = 50 µm in c and 10 µm in d;e & f, Representative images of Nrl.Cre+/-/TdTomato+/- chimera after receiving a subretinal injection of AAV-Nrl.Cre. The injected eyes show widespread recombination, which also shows the anticipated mosaic stripe patterning; Scale bar = 50 µm in (e) and 10 µm in (f);


figf1 ( a ) MDCK / pGFP - LC3 cells infected with a wild - type or ΔactA2 strain of L . monocytogenes were stained with an anti - Listeria antibody ( red ) and rhodamine phalloidin ( stains F - actin ; light blue ) . Scale bars , 10 μm . Arrowheads indicate GFP - LC3 - positive bacteria . ( b ) High magnification of framed images in a , and graphs show intensity of the fluoresence signal along the arrow . ( c ) Quantification of the number of GFP - LC3 - positive bacteria shown in a . Data are mean ± s . e . m . At least 500 bacteria were counted in each experiment ( n = 7 and 6 experiments at 2 and 4 h , respectively ) . ( d ) LC3 levels in infected MDCK cells . MDCK cells were infected with wild - type or ΔactA2 bacteria for the indicated times . EBSS ( Earle ' s balanced salt solution , a starvation medium ) and rapamycin ( 100 μg ml − 1 ) were used as controls . ( e - f ) Intracellular survival of wild - type or ΔactA2 bacteria in MDCK cells ( e ; n = 8 ) , MDCK cells treated with 10 mM 3 - MA ( f ; n = 4 ) , ( e - h ) Intracellular survival of wild - type or ΔactA2 bacteria in atg5 + / + MEF cells ( g ; n = 8 and 7 for wild - type and ΔactA2 , respectively ) or atg5 − / − MEF cells ( h ; n = 4 and 3 for wild - type and ΔactA2 , respectively ) . Data are presented as the number of colony - forming units ( CFU ) relative to that at 30 min after infection , and are mean ± s . e . m . WT , wild type .

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 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 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 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

figf1(a) MDCK/pGFP-LC3 cells infected with a wild-type or ΔactA2 strain of L. monocytogenes were stained with an anti-Listeria antibody (red) and rhodamine phalloidin (stains F-actin; light blue). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive bacteria. (b) High magnification of framed images in a, and graphs show intensity of the fluoresence signal along the arrow. (c) Quantification of the number of GFP-LC3-positive bacteria shown in a. Data are mean ± s.e.m. At least 500 bacteria were counted in each experiment (n = 7 and 6 experiments at 2 and 4 h, respectively).(d) LC3 levels in infected MDCK cells. MDCK cells were infected with wild-type or ΔactA2 bacteria for the indicated times. EBSS (Earle's balanced salt solution, a starvation medium) and rapamycin (100 μg ml−1) were used as controls.(e-f) Intracellular survival of wild-type or ΔactA2 bacteria in MDCK cells (e; n = 8), MDCK cells treated with 10 mM 3-MA (f; n = 4),(e-h) Intracellular survival of wild-type or ΔactA2 bacteria in atg5+/+ MEF cells (g; n = 8 and 7 for wild-type and ΔactA2, respectively) or atg5−/− MEF cells (h; n = 4 and 3 for wild-type and ΔactA2, respectively). Data are presented as the number of colony-forming units (CFU) relative to that at 30 min after infection, and are mean ± s.e.m. WT, wild type.


figf2 ( b , c ) Quantification of the number of GFP - LC3 - positive bacteria ( b ) or actin - accumulating bacteria ( c ) . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 3 experiments ) . ( d ) Colocalization of ubiquitin with ΔactA2 . MDCK / pGFP - LC3 cells infected with ΔactA2 bacteria were stained with an anti - Listeria antibody ( blue ) and anti - ubiquitin antibody ( red ) . Scale bars , 10 μm . Arrowheads indicate GFP - LC3 - positive or ubiquitin - positive bacteria . ( e ) High magnification of framed image in d , and the graph shows the intensity of the fluorescence signal along the arrow . ( f ) Quantification of the number of GFP - LC3 - positive , ubiquitin - positive or GFP - LC3 - and ubiquitin - positive bacteria . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 3 experiments ) . ( g ) Immunogold electron micrograph of MDCK / pGFP - LC3 cells infected with ΔactA2 . Gold particles of 5 nm ( arrowheads ) indicate GFP - LC3 , and gold particles of 10 nm ( arrows ) indicate ubiquitin . Scale bars , 0 . 5 μm . ( h ) Kinetics of the generation of GFP - LC3 - positive or ubiquitin - positive bacteria . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 3 experiments ) . * P 0 . 001 . ( i ) Quantification of the number of ubiquitin - positive or GFP - LC3 - positive bacteria . MDCK / pGFP - LC3 cells infected with wild - type or ΔactA2 bacteria were untreated or treated with 3 - MA ( 10 mM ) . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n ≥ 3 experiments ) . Ub , ubiquitin . WT , wild type .

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

figf2(b, c) Quantification of the number of GFP-LC3-positive bacteria (b) or actin-accumulating bacteria (c). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments).(d) Colocalization of ubiquitin with ΔactA2. MDCK/pGFP-LC3 cells infected with ΔactA2 bacteria were stained with an anti-Listeria antibody (blue) and anti-ubiquitin antibody (red). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive or ubiquitin-positive bacteria. (e) High magnification of framed image in d, and the graph shows the intensity of the fluorescence signal along the arrow. (f) Quantification of the number of GFP-LC3-positive, ubiquitin-positive or GFP-LC3- and ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments).(g) Immunogold electron micrograph of MDCK/pGFP-LC3 cells infected with ΔactA2. Gold particles of 5 nm (arrowheads) indicate GFP-LC3, and gold particles of 10 nm (arrows) indicate ubiquitin. Scale bars, 0.5 μm.(h) Kinetics of the generation of GFP-LC3-positive or ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). *P 0.001. (i) Quantification of the number of ubiquitin-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3 cells infected with wild-type or ΔactA2 bacteria were untreated or treated with 3-MA (10 mM). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n ≥ 3 experiments). Ub, ubiquitin. WT, wild type.


figf3 ( a ) Quantification of the number of ubiquitin - positive , p62 - positive or GFP - LC3 - positive bacteria . MDCK / pGFP - LC3 / pp62 - 3 × Myc cells were infected with a wild - type or ΔactA2 strain of L . monocytogenes for 2 h . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 6 experiments ) . ( b ) Colocalization of p62 with ΔactA2 . MDCK / pGFP - LC3 / pp62 - 3 × Myc cells were stained with an anti - ubiquitin antibody ( light blue ) , anti - Myc antibody ( red ) , DAPI ( white ) and anti - GFP - LC3 ( green ) . Scale bar , 10 μm . Arrowheads indicate GFP - LC3 - , ubiquitin - or p62 - positive bacteria . ( c ) Quantification of the number of ubiquitin - positive , p62 - positive or GFP - LC3 - positive bacteria . p62 − / − / p62 cells were infected with ΔactA2 bacteria for 2 h . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 6 and 3 experiments for p62 − / − and p62 − / − / p62 , respectively ) ( d , e ) Intracellular survival of wild - type or ΔactA2 bacteria in p62 − / − cells ( d ) or p62 − / − / p62 cells ( e ; colony - forming units , CFU , relative to CFU 30 min after infection ) . Data are presented as mean ± s . e . m . ( n ≥ 4 experiments ) . ( g ) Generation of p62 - 3 × Myc mutants in p62 − / − cells . ( h ) Quantification of the number of GFP - positive or GFP - LC3 - positive bacteria . p62 − / − / p62 ( wild type , ΔLIR , ΔUBA , ΔLIR - ΔUBA or K7AD69A ) cells were infected with ΔactA2 bacteria and stained with anti - bacteria and anti - Myc antibodies ( Supplementary Information , Fig . S4e ) . Data are mean ± s . e . m . , at least 500 LC3 were counted in each experiment ( n = 3 experiments ) . P 0 . 001 versus p62 - positive LC3 in p62 − / − / p62 wild - type cells , P 0 . 001 versus GFP - p62 - positive bacteria in p62 − / − / ubiquitinwild - type cells . Ub , ubiquitin . WT , wild type . A full scan of g is shown in Supplementary Information , Fig . S7 .

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 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 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 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 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

figf3(a) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3/pp62-3×Myc cells were infected with a wild-type or ΔactA2 strain of L. monocytogenes for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 experiments).(b) Colocalization of p62 with ΔactA2. MDCK/pGFP-LC3/pp62-3×Myc cells were stained with an anti-ubiquitin antibody (light blue), anti-Myc antibody (red), DAPI (white) and anti-GFP-LC3 (green). Scale bar, 10 μm. Arrowheads indicate GFP-LC3-, ubiquitin- or p62-positive bacteria.(c) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. p62−/−/p62 cells were infected with ΔactA2 bacteria for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 and 3 experiments for p62−/− and p62−/−/p62, respectively)(d, e) Intracellular survival of wild-type or ΔactA2 bacteria in p62−/− cells (d) or p62−/−/p62 cells (e; colony-forming units, CFU, relative to CFU 30 min after infection). Data are presented as mean ± s.e.m. (n ≥ 4 experiments).(g) Generation of p62-3×Myc mutants in p62−/− cells.(h) Quantification of the number of GFP-positive or GFP-LC3-positive bacteria. p62−/−/p62 (wild type, ΔLIR, ΔUBA, ΔLIR-ΔUBA or K7AD69A) cells were infected with ΔactA2 bacteria and stained with anti-bacteria and anti-Myc antibodies (Supplementary Information, Fig. S4e). Data are mean ± s.e.m., at least 500 LC3 were counted in each experiment (n = 3 experiments). P 0.001 versus p62-positive LC3 in p62−/−/p62 wild-type cells, P 0.001 versus GFP-p62-positive bacteria in p62−/−/ubiquitinwild-type cells. Ub, ubiquitin. WT, wild type. A full scan of g is shown in Supplementary Information, Fig. S7.


figf4 ( b - e ) The series of pEGFP - ActA - Q79C constructs was transfected into COS - 7 cells . Cells were fixed with 4 % Paraformaldehyde after 18 h and stained with an anti - Arp2 antibody ( b ) , an anti - VASP antibody ( c ) , an anti - ubiquitin antibody ( d ) or an anti - p62 antibody ( e ; red ) . Scale bars , 10 μm . Arrowheads indicate Arp2 - , VASP - , ubiquitin - or p62 - positive Q79C . Ub , ubiquitin .

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

figf4(b-e) The series of pEGFP-ActA-Q79C constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 18 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive Q79C. Ub, ubiquitin.


figf5 ( b - e ) The series of pEGFP - ActA - 170 constructs was transfected into COS - 7 cells . Cells were fixed with 4 % Paraformaldehyde after 14 h and stained with an anti - Arp2 antibody ( b ) , an anti - VASP antibody ( c ) , an anti - ubiquitin antibody ( d ) or an anti - p62 antibody ( e ; red ) . Scale bars , 10 μm . Arrowheads indicate Arp2 - , VASP - , ubiquitin - or p62 - positive 170 . Ub , ubiquitin .

figf5(b-e) The series of pEGFP-ActA-170 constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 14 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive 170. Ub, ubiquitin.


figf1 ( b ) PC12 cells transfected with either 60Q or 60Q - HSC70bm . The presence of two HSC70bm motifs in 60Q - HSC70bm reduced the number of inclusions as compared to 60Q ( scale bar , 20 μm ) . ( c ) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q - HSC70bm . 10 mM 3 - methyladenine ( 3 - MA ) , 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy , the proteases , cathepsins D and E , and cathepsin A , respectively . Bars in c represent relative mean values ± s . e . m . from four independent experiments , with levels of aggregation observed for 60Q normalized to a value of 1 . Quantifications were performed by ArrayScan .

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

figf1(b) PC12 cells transfected with either 60Q or 60Q-HSC70bm. The presence of two HSC70bm motifs in 60Q-HSC70bm reduced the number of inclusions as compared to 60Q (scale bar, 20 μm). (c) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q-HSC70bm. 10 mM 3-methyladenine (3-MA), 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy, the proteases, cathepsins D and E, and cathepsin A, respectively. Bars in c represent relative mean values ± s.e.m. from four independent experiments, with levels of aggregation observed for 60Q normalized to a value of 1. Quantifications were performed by ArrayScan.


figf2 ( b ) Confocal images of the 150Q Neuro2a cells transfected with R , RH , RS , RHS , RQ and RHQ . Molecules containing QBP1 co - localized with the tNHTT - 150Q - EGFP inclusions ( bar , 5 μm ) . Cells with inclusions were chosen to illustrate the co - localization of QBP1 - containing molecules with tNHTT - 150Q - EGFP . ( c ) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT - 150Q - EGFP on the gel top . Note the shift of tNHTT - 150Q - EGFP from an insoluble to a soluble form when RQ was co - expressed . The levels of tNHTT - 16Q - EGFP were not affected . Full - length western blots are presented in Supplementary Figure 13 . ( d ) RHQ reduced the levels of aggregated and soluble forms of tNHTT - 150Q - EGFP by 81 . 8 % and 33 . 6 % , respectively . WB , western blot . ( e ) Quantification of tNHTT - 16Q - EGFP protein levels . ( f ) ArrayScan analysis revealed 28 % and 56 % reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ , respectively , after 24 h of differentiation and induction . ( g ) In 150Q - nls Neuro2a cells , expression of RQ and RHQ reduced the number of tNHTT - 150Q - nls - EGFP inclusion by 23 % ( RQ ) and 53 % ( RHQ ) after 48 h of differentiation and induction compared to RS and RHS , respectively . Bars in d and e represent mean values ± s . e . m . from four independent experiments . Quantifications were performed by ArrayScan .

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 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 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

figf2(b) Confocal images of the 150Q Neuro2a cells transfected with R, RH, RS, RHS, RQ and RHQ. Molecules containing QBP1 co-localized with the tNHTT-150Q-EGFP inclusions (bar, 5 μm). Cells with inclusions were chosen to illustrate the co-localization of QBP1-containing molecules with tNHTT-150Q-EGFP.(c) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT-150Q-EGFP on the gel top. Note the shift of tNHTT-150Q-EGFP from an insoluble to a soluble form when RQ was co-expressed. The levels of tNHTT-16Q-EGFP were not affected. Full-length western blots are presented in Supplementary Figure 13.(d) RHQ reduced the levels of aggregated and soluble forms of tNHTT-150Q-EGFP by 81.8% and 33.6%, respectively. WB, western blot. (e) Quantification of tNHTT-16Q-EGFP protein levels.(f) ArrayScan analysis revealed 28% and 56% reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ, respectively, after 24 h of differentiation and induction. (g) In 150Q-nls Neuro2a cells, expression of RQ and RHQ reduced the number of tNHTT-150Q-nls-EGFP inclusion by 23% (RQ) and 53% (RHQ) after 48 h of differentiation and induction compared to RS and RHS, respectively. Bars in d and e represent mean values ± s.e.m. from four independent experiments. Quantifications were performed by ArrayScan.


figf3 ( a ) Western blot analysis of the chase experiment in 60Q Neuro2a cells . Full - length western blots are presented in Supplementary Figure 13 . ( b ) RHQ enhanced the tNHTT - 60Q - EGFP degradation during the chase period . 5 μM MG132 , 10 mM 3 - methyladenine and 15 mM ammonium chloride ( NH4Cl ) were used to inhibit proteasome , macroautophagy and lysosomal degradation , respectively . NH4Cl had no effect on proteasome activity and did not affect cell viability ( Supplementary Fig . 4a , b ) . Bars in b represent relative mean values ± s . e . m . from three independent experiments . Protein levels at time 0 represent the control condition ( arbitrarily set as 1 ) .

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

figf3(a) Western blot analysis of the chase experiment in 60Q Neuro2a cells. Full-length western blots are presented in Supplementary Figure 13. (b) RHQ enhanced the tNHTT-60Q-EGFP degradation during the chase period. 5 μM MG132, 10 mM 3-methyladenine and 15 mM ammonium chloride (NH4Cl) were used to inhibit proteasome, macroautophagy and lysosomal degradation, respectively. NH4Cl had no effect on proteasome activity and did not affect cell viability (Supplementary Fig. 4a,b). Bars in b represent relative mean values ± s.e.m. from three independent experiments. Protein levels at time 0 represent the control condition (arbitrarily set as 1).


figf4 ( a , b ) Constructs identical to those described in Figure 2a , but lacking mRFP ( representations of S , HS , Q , HQ are shown in Supplementary Fig . 3a ) were co - transfected with tNHTT - 16Q - EGFP ( a ) and tNHTT - 150Q - EGFP ( b ) constructs into Neuro2a cells ( bar , 4 μm ) . ( c ) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co - transfected with tNHTT - 60Q - EGFP and R , RS , RHS , RQ or RHQ . PNS , post - nuclear supernatant . ( d ) Quantification of HTT translocation into lysosomes . Levels of HTT detected by EM48 ( anti - huntingtin antibody ) were normalized to levels of β - tubulin ( in PNS ) and levels of cathepsin D ( in lysosomes ) . ( e ) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R , RS , RHS , RQ and RHQ and shRNA specific for HSC70 and / or Lamp2a . ( blue , Hoechst 33258 ; green , tNHTT - 150Q - EGFP ; red , tested molecule ) . ( f ) RNAi of HSC70 , Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT - 150Q - EGFP inclusions . ( g ) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and / or Lamp2a . Full - length western blots are presented in Supplementary Figure 13 . ( h ) Silencing of HSC70 , Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT - 150Q - EGFP . Bars in d and f represent mean values ± s . e . m . from three independent experiments . Bars in h represent relative mean values ± s . e . m . from three independent experiments . For h , values for the R construct were arbitrarily set to 1 .

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 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 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 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 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 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

figf4(a,b) Constructs identical to those described in Figure 2a, but lacking mRFP (representations of S, HS, Q, HQ are shown in Supplementary Fig. 3a) were co-transfected with tNHTT-16Q-EGFP (a) and tNHTT-150Q-EGFP (b) constructs into Neuro2a cells (bar, 4 μm).(c) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co-transfected with tNHTT-60Q-EGFP and R, RS, RHS, RQ or RHQ. PNS, post-nuclear supernatant.(d) Quantification of HTT translocation into lysosomes. Levels of HTT detected by EM48 (anti-huntingtin antibody) were normalized to levels of β-tubulin (in PNS) and levels of cathepsin D (in lysosomes).(e) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R, RS, RHS, RQ and RHQ and shRNA specific for HSC70 and/or Lamp2a. (blue, Hoechst 33258; green, tNHTT-150Q-EGFP; red, tested molecule).(f) RNAi of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT-150Q-EGFP inclusions.(g) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and/or Lamp2a. Full-length western blots are presented in Supplementary Figure 13.(h) Silencing of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT-150Q-EGFP. Bars in d and f represent mean values ± s.e.m. from three independent experiments. Bars in h represent relative mean values ± s.e.m. from three independent experiments. For h, values for the R construct were arbitrarily set to 1.


figf5 ( a ) Filter trap assay analysis of HTT - polyQaggregation in nine R6 / 2 mouse brains injected with three different combinations of rAAV . ( b ) Quantification of the SDS - insoluble material detected by filter trap assay ( * , P = 0 . 037 ; * * , P = 0 . 0083 ; * * * , P = 0 . 0015 ) . ( c ) Western blot analysis of HTT aggregation ( gel top , EM48 antibody ) , soluble HTT ( 1C2 antibody ) , and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates ( AGERA ) . Full - length western blots are presented in Supplementary Figure 13 . ( d ) Quantification of the gel top - detected aggregation ( * , P = 0 . 093 ; * * , P = 0 . 0027 ; * * * , P = 0 . 0045 ) . ( e ) Quantification of soluble HTT - polyQ ( * , P = 0 . 034 ; * * , P = 0 . 00003 ; * * * , P = 0 . 0001 ) . ( f ) Brain sections stained with anti - RFP , EM48 and anti - ubiquitin antibodies ( bar , 40 μm ) . ( g ) Numbers of ubiquitin - positive inclusions in the transduced areas ( * , P = 0 . 0026 ; * * , P = 5 . 535 × 10 − 8 ; * * * , P = 2 . 009 × 10 − 8 ) . Bars in b , d , e and g represent the relative mean values ± s . e . m . from three brains injected with different rAAV in each striatum . R in R / Q and R / HQ , and Q in Q / HQ brains represent the control value of 1 .

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 O O O O 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

figf5(a) Filter trap assay analysis of HTT-polyQaggregation in nine R6/2 mouse brains injected with three different combinations of rAAV. (b) Quantification of the SDS-insoluble material detected by filter trap assay (*, P = 0.037; **, P = 0.0083; ***, P = 0.0015).(c) Western blot analysis of HTT aggregation (gel top, EM48 antibody), soluble HTT (1C2 antibody), and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates (AGERA). Full-length western blots are presented in Supplementary Figure 13. (d) Quantification of the gel top-detected aggregation (*, P = 0.093; **, P = 0.0027; ***, P = 0.0045). (e) Quantification of soluble HTT-polyQ (*, P = 0.034; **, P = 0.00003; ***, P = 0.0001).(f) Brain sections stained with anti-RFP, EM48 and anti-ubiquitin antibodies (bar, 40 μm). (g) Numbers of ubiquitin-positive inclusions in the transduced areas (*, P = 0.0026; **, P = 5.535 × 10−8; ***, P = 2.009 × 10−8). Bars in b, d, e and g represent the relative mean values ± s.e.m. from three brains injected with different rAAV in each striatum. R in R/Q and R/HQ, and Q in Q/HQ brains represent the control value of 1.


figf6 ( a ) Relative body weight changes in mice after bilateral injection with rAAV - R , rAAV - Q and rAAV - HQ at 6 - 14 weeks of age ( n = 11 for all three groups until 12 weeks ; at 14 weeks , n = 7 for R / R , n = 10 for Q / Q , n = 11 for HQ / HQ ) ( * , P = 0 . 0401 ; * * , P = 0 . 0081 ; * * * , P = 0 . 0182 ; * * * * , P = 0 . 0225 ) . ( b ) Clasping scores . n = 11 for all three groups ( scale : 0 , no clasping ; 1 , clasping of the forelimbs only ; 2 , clasping of both fore and hind limbs once or twice ; 3 , clasping of both fore and hind limbs more than 3 times or more than 5 s ) . ( c ) Rotarod performance ; n = 11 for all three groups ( * , P = 0 . 0055 ; * * , P = 0 . 002 ; * * * , P = 0 . 0455 ; * * * * , P = 0 . 0242 ) . ( d ) Survival curves for mice after bilateral injection with rAAV - R , rAAV - Q , and rAAV - HQ ; n = 11 for all groups . Median survival ( days ) : R / R - 108 ; Q / Q - 123 ; HQ / HQ - 140 . Mean survival ( days ) : R / R - 105 . 5 ± 3 . 9 ; Q / Q - 122 . 3 ± 4 . 8 ; HQ / HQ - 136 . 2 ± 4 . 4 . Log - rank test , P 0 . 0001 ; Wilcoxon test , P 0 . 0001 .

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figf6(a) Relative body weight changes in mice after bilateral injection with rAAV-R, rAAV-Q and rAAV-HQ at 6-14 weeks of age (n = 11 for all three groups until 12 weeks; at 14 weeks, n = 7 for R/R, n = 10 for Q/Q, n = 11 for HQ/HQ) (*, P = 0.0401; **, P = 0.0081; ***, P = 0.0182; ****, P = 0.0225).(b) Clasping scores. n = 11 for all three groups (scale: 0, no clasping; 1, clasping of the forelimbs only; 2, clasping of both fore and hind limbs once or twice; 3, clasping of both fore and hind limbs more than 3 times or more than 5 s).(c) Rotarod performance; n = 11 for all three groups (*, P = 0.0055; **, P = 0.002; ***, P = 0.0455; ****, P = 0.0242).(d) Survival curves for mice after bilateral injection with rAAV-R, rAAV-Q, and rAAV-HQ; n = 11 for all groups. Median survival (days): R/R-108; Q/Q-123; HQ/HQ-140. Mean survival (days): R/R-105.5 ± 3.9; Q/Q-122.3 ± 4.8; HQ/HQ-136.2 ± 4.4.Log-rank test, P 0.0001; Wilcoxon test, P 0.0001.


Figure 1 ( A ) HeLa cells were incubated for 4 h in either EBSS or media without glucose / pyruvate . Lysates were then analysed by western blot for the presence of MCL ‐ 1 , as well as actin as a loading control . ( B ) CGNs were subjected to 17 h hypoxia after 7 daysin vitro . Protein lysates were collected at indicated times after reoxygenation and blotted for MCL ‐ 1 expression and for actin as a loading control . ( C ) H1299 cells stably expressing GFP - LC3 were incubated for 2 h in EBSS , and expression of various BCL ‐ 2 homologues was analysed by western blot . Alternatively , primary cortical neurons were incubated for 4 h in media without glucose / pyruvate . ( D ) H1299 cells stably expressing GFP - LC3 were incubated for 3 h in media without glucose / pyruvate , followed by re ‐ addition of glucose and pyruvate for the indicated times . ( E ) HeLa cells were infected with 5 p . f . u . per cell of either Ad HA ‐ tBID or Ad LacZ for 7 h , the media being changed to EBSS for the last 3 h where indicated . Alternatively , cells were treated with 50 μM camptothecin for 3 h . ( F , G ) HeLa cells were treated as in E in the presence of 50 μM zVAD ‐ FMK and analysed by immunofluorescence microscopy for active BAX ( 6A7 epitope ; F ) or cyt c ( G ) . Data are expressed as the average of three experiments ± s . d . * P0 . 01 .

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 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 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 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 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

Figure 1(A) HeLa cells were incubated for 4 h in either EBSS or media without glucose/pyruvate. Lysates were then analysed by western blot for the presence of MCL‐1, as well as actin as a loading control.(B) CGNs were subjected to 17 h hypoxia after 7 daysin vitro. Protein lysates were collected at indicated times after reoxygenation and blotted for MCL‐1 expression and for actin as a loading control.(C) H1299 cells stably expressing GFP-LC3 were incubated for 2 h in EBSS, and expression of various BCL‐2 homologues was analysed by western blot. Alternatively, primary cortical neurons were incubated for 4 h in media without glucose/pyruvate.(D) H1299 cells stably expressing GFP-LC3 were incubated for 3 h in media without glucose/pyruvate, followed by re‐addition of glucose and pyruvate for the indicated times.(E) HeLa cells were infected with 5 p.f.u. per cell of either Ad HA‐tBID or Ad LacZ for 7 h, the media being changed to EBSS for the last 3 h where indicated. Alternatively, cells were treated with 50 μM camptothecin for 3 h.(F, G) HeLa cells were treated as in E in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence microscopy for active BAX (6A7 epitope; F) or cyt c (G). Data are expressed as the average of three experiments±s.d. *P0.01.


Figure 2 ( A ) H1299 cells stably expressing GFP - LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD ‐ FMK and analysed by immunofluorescence for cyt c or active BAX ( 6A7 epitope ) . As a positive control , cells were infected with 5 p . f . u . per cell Ad HA ‐ tBID for 7 h . Data are expressed as the average of three experiments ± s . d . ( B ) H1299 cells stably expressing GFP - LC3 were incubated for 4 h in either EBSS or media without glucose / pyruvate . Cells were then fixed , stained with an antibody against cyt c and analysed by immunofluorescence . Scale bars = 50 μm . ( Alternatively ( C ) , the cells were analysed by western blot for the presence of MCL ‐ 1 and the autophagic markers GFP - LC3 ( anti ‐ GFP antibody ) and p62 . ( D ) H1299 cells stably expressing GFP - LC3 were incubated for the indicated times in either EBSS or media without glucose / pyruvate and analysed by western blot for the presence of MCL ‐ 1 and autophagic markers . ( E , F ) H1299 cells stably expressing GFP - LC3 were pre ‐ incubated for 1 h with 5 mM 3MA ( E ) or 200 nM bafilomycin ( F ) , and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin . Lysates were then analysed for the presence of MCL ‐ 1 , the autophagy marker p62 and actin as a loading control .

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Figure 2(A) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence for cyt c or active BAX (6A7 epitope). As a positive control, cells were infected with 5 p.f.u. per cell Ad HA‐tBID for 7 h. Data are expressed as the average of three experiments±s.d.(B) H1299 cells stably expressing GFP-LC3 were incubated for 4 h in either EBSS or media without glucose/pyruvate. Cells were then fixed, stained with an antibody against cyt c and analysed by immunofluorescence. Scale bars=50 μm.( Alternatively (C), the cells were analysed by western blot for the presence of MCL‐1 and the autophagic markers GFP-LC3 (anti‐GFP antibody) and p62.(D) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in either EBSS or media without glucose/pyruvate and analysed by western blot for the presence of MCL‐1 and autophagic markers.(E, F) H1299 cells stably expressing GFP-LC3 were pre‐incubated for 1 h with 5 mM 3MA (E) or 200 nM bafilomycin (F), and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin. Lysates were then analysed for the presence of MCL‐1, the autophagy marker p62 and actin as a loading control.


Figure 3 ( A ) H1299 cells stably expressing GFP - LC3 were infected with 10 p . f . u . per cell of either Ad LacZ or Ad Myc ‐ MCL ‐ 1 for 24 h then incubated in EBSS for 4 h . Alternatively , cells were transfected with control or MCL ‐ 1 siRNA 24 h before EBSS treatment . Cells were stained with an antibody against cyt c and analysed by immunofluorescence . Representative images are shown . Scale bars = 50 μm . ( B ) The percent of the cell surface area covered by GFP - LC3 vesicles was quantified using the Image J software . n > 70 cells in three experiment , * P0 . 01 . ( C , D ) H1299 cells stably expressing GFP - LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated . The levels of endogenous LC3 ‐ II relative to actin were quantified by western blot in C . Data are expressed as the average of three experiments ± s . d , * P0 . 05 . ( E , F ) H1299 cells stably expressing GFP - LC3 were treated as in A and analysed by western blot for the presence of p62 and GFP - LC3 . ( G ) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of MCL ‐ 1 and p62 by western blot 3 days later . ( H ) Transformed wild ‐ type ( WT ) and MCL ‐ 1Δ / Δ ( KO ) MEFs were analysed by western blot for the expression of endogenous LC3 . ( I , J ) Colocalization of GFP - LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunofluorescence in cells treated as in A . The number of LC3 ‐ positive vesicles that colocalised with LAMP1 was quantified in I . Data are expressed as the percentage of LC3 vesicles per cell that are LAMP1 ‐ positive ± s . e . m . , with at least 14 cells per condition . Representative images are shown in J . Arrows indicate LC3 ‐ positive vesicles colocalizing with LAMP1 . Scale bars = 10 μm .

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 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 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 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 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 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

Figure 3(A) H1299 cells stably expressing GFP-LC3 were infected with 10 p.f.u. per cell of either Ad LacZ or Ad Myc‐MCL‐1 for 24 h then incubated in EBSS for 4 h. Alternatively, cells were transfected with control or MCL‐1 siRNA 24 h before EBSS treatment. Cells were stained with an antibody against cyt c and analysed by immunofluorescence. Representative images are shown. Scale bars=50 μm.(B) The percent of the cell surface area covered by GFP-LC3 vesicles was quantified using the Image J software. n>70 cells in three experiment, *P0.01.(C, D) H1299 cells stably expressing GFP-LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated. The levels of endogenous LC3‐II relative to actin were quantified by western blot in C. Data are expressed as the average of three experiments±s.d, *P0.05.(E, F) H1299 cells stably expressing GFP-LC3 were treated as in A and analysed by western blot for the presence of p62 and GFP-LC3.(G) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of MCL‐1 and p62 by western blot 3 days later.(H) Transformed wild‐type (WT) and MCL‐1Δ/Δ (KO) MEFs were analysed by western blot for the expression of endogenous LC3.(I, J) Colocalization of GFP-LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunofluorescence in cells treated as in A. The number of LC3‐positive vesicles that colocalised with LAMP1 was quantified in I. Data are expressed as the percentage of LC3 vesicles per cell that are LAMP1‐positive±s.e.m., with at least 14 cells per condition. Representative images are shown in J. Arrows indicate LC3‐positive vesicles colocalizing with LAMP1. Scale bars=10 μm.


Figure 4 ( A ) MCL ‐ 1 interacts with Beclin ‐ 1 . Lysates from H1299 stably expressing GFP - LC3 were immunoprecipitated using control or MCL ‐ 1 ‐ specific antibodies and analysed for the presence of Beclin ‐ 1 by western blot . ( B ) H1299 cells stably expressing GFP - LC3 were fractionated into HM ( containing mitochondria ) , and LM ( containing ER ) . Fractions were analysed for the presence of MCL ‐ 1 , BCL ‐ 2 , Beclin ‐ 1 , the mitochondrial marker mtHSP70 and the ER marker calreticulin . ( C ) MCL ‐ 1 was immunoprecipitated from HM and LM before analysing the presence of MCL ‐ 1 and Beclin ‐ 1 .

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 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

Figure 4(A) MCL‐1 interacts with Beclin‐1. Lysates from H1299 stably expressing GFP-LC3 were immunoprecipitated using control or MCL‐1‐specific antibodies and analysed for the presence of Beclin‐1 by western blot.(B) H1299 cells stably expressing GFP-LC3 were fractionated into HM (containing mitochondria), and LM (containing ER). Fractions were analysed for the presence of MCL‐1, BCL‐2, Beclin‐1, the mitochondrial marker mtHSP70 and the ER marker calreticulin.(C) MCL‐1 was immunoprecipitated from HM and LM before analysing the presence of MCL‐1 and Beclin‐1.


Figure 5 ( A ) Control and MCL ‐ 1Δ / Δ mice . ( B ) Expression levels of various BCL ‐ 2 homologues in the cortices of animals of the indicated genotypes . ( C ) Brain morphology of MCL ‐ 1Δ / Δ . Brain sections were stained with cresyl violet and imaged using a × 1 objective . ( D - G ) Cortical neurons around the lesion in MCL ‐ 1Δ / Δ animals are positive for the autophagic marker LC3 . Brain sections from animals with the indicated genotypes ( P14 for E , G ; P7 for H ) were stained for LC3 ( Green ) and the neuronal marker NeuN ( in H , red ) , along with the nuclear stain Hoechst ( Blue ) . Confocal images were taken using × 10 ( E ) or × 63 ( G , H ) objectives . Scale bars = 200 μm ( E ) ; 50 μm ( G , H ) . Quantification of the LC3 staining is shown in D . The total number of cells was determined by counting the Hoechst ‐ positive nuclei . Data are expressed as percent of LC3 ‐ positive cells in at least three animals per genotype ± s . d . * P0 . 01 . ( F ) Quantification of the total surface area occupied by autophagosomes . Data are expressed as percent of total cortical area covered by autophagosomes in at least 16 EM images per genotype ± s . d . P0 . 005 ( I ) Representative EM images from MCL ‐ 1 + / Δ and MCL ‐ 1Δ / ΔP14mice . N , nucleus ; * , autophagosomal structures ; arrowheads , double membrane ; arrow , mitochondrion inside a vesicle .

O O B-PANEL_START 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 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 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

Figure 5(A) Control and MCL‐1Δ/Δ mice.(B) Expression levels of various BCL‐2 homologues in the cortices of animals of the indicated genotypes.(C) Brain morphology of MCL‐1Δ/Δ. Brain sections were stained with cresyl violet and imaged using a × 1 objective.(D-G) Cortical neurons around the lesion in MCL‐1Δ/Δ animals are positive for the autophagic marker LC3. Brain sections from animals with the indicated genotypes (P14 for E, G; P7 for H) were stained for LC3 (Green) and the neuronal marker NeuN (in H, red), along with the nuclear stain Hoechst (Blue). Confocal images were taken using × 10 (E) or × 63 (G, H) objectives. Scale bars=200 μm (E); 50 μm (G, H). Quantification of the LC3 staining is shown in D. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of LC3‐positive cells in at least three animals per genotype±s.d. *P0.01. (F) Quantification of the total surface area occupied by autophagosomes. Data are expressed as percent of total cortical area covered by autophagosomes in at least 16 EM images per genotype±s.d. P0.005 (I) Representative EM images from MCL‐1+/Δ and MCL‐1Δ/ΔP14mice. N, nucleus; *, autophagosomal structures; arrowheads, double membrane; arrow, mitochondrion inside a vesicle.


Figure 6 ( A ) Cortices from the indicated genotypes and ages were analysed by western blot for the presence of active caspase ‐ 3 , p62 and MCL ‐ 1 . Primary cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase ‐ 3 . ( B ) Active BAX ( 6A7 ) and active caspase ‐ 3 were also quantified by staining the cortex of 1 ‐ to 14 ‐ day ‐ old MCL ‐ 1Δ / Δ mice . Representative images from at least three sections from each animal were counted . The total number of cells was determined by counting the Hoechst ‐ positive nuclei . Data are expressed as percent of positive cells for at least three animals per genotype ± s . d . * P0 . 05 . ( C ) Active BAX is present in a subset of LC3 ‐ positive cells . Sections from P7 and P14 MCL ‐ 1Δ / Δ brains were stained for LC3 ( Green ) and active BAX ( 6A7 ; Red ) . Confocal images were taken using × 63 objective . Arrows point to BAX ‐ positive , LC3 ‐ negative cells . Representative images are shown . Scale bars = 50 μm . Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of the caspase ‐ cleaved fragment of the caspase ‐ 3 substrate CAS5 days later . Alternatively ( E ) , the neurons were analysed for cytochrome c release by immunofluorescence . Data are expressed as percent of positive cells for at least three embryos per genotype ± s . 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 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 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

Figure 6(A) Cortices from the indicated genotypes and ages were analysed by western blot for the presence of active caspase‐3, p62 and MCL‐1. Primary cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase‐3.(B) Active BAX (6A7) and active caspase‐3 were also quantified by staining the cortex of 1‐ to 14‐day‐old MCL‐1Δ/Δ mice. Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for at least three animals per genotype±s.d. *P0.05.(C) Active BAX is present in a subset of LC3‐positive cells. Sections from P7 and P14 MCL‐1Δ/Δ brains were stained for LC3 (Green) and active BAX (6A7; Red). Confocal images were taken using × 63 objective. Arrows point to BAX‐positive, LC3‐negative cells. Representative images are shown. Scale bars=50 μm.Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate CAS5 days later. Alternatively (E), the neurons were analysed for cytochrome c release by immunofluorescence. Data are expressed as percent of positive cells for at least three embryos per genotype±s.d.