PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed ( F ) cells lacking expression of analyzed lineage markers ( Lin - ) .

PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (F) cells lacking expression of analyzed lineage markers (Lin-).


Analysis of frequency of colonies derived from HO - 1 + / + or from HO - 1 - / - MSC co - cultures among identified clusters . Enrichment in cluster analyzed by Fisher exact test , * * p < 0 . 01 , two - tailed unpaired t - test .

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Analysis of frequency of colonies derived from HO-1+/+ or from HO-1-/- MSC co-cultures among identified clusters. Enrichment in cluster analyzed by Fisher exact test, **p<0.01, two-tailed unpaired t-test.


Sdf1α mRNA levels in BM of HO - 1 + / + or HO - 1 - / - mice after administration of Noggin . Data are shown as mean ± SEM . * p < 0 . 05 , two - tailed unpaired t - test , n = 10 /

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Sdf1α mRNA levels in BM of HO-1+/+ or HO-1-/- mice after administration of Noggin. Data are shown as mean ± SEM. *p< 0.05, two-tailed unpaired t-test, n = 10/group


LT - HSC frequency of LT - HSCs after administration of Noggin to HO - 1 + / + or HO - 1 - / - mice . Data are shown as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , two - tailed unpaired t - test , n = 8 - 10 /

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LT-HSC frequency of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice. Data are shown as mean ± SEM. *p< 0.05, **p<0.01, two-tailed unpaired t-test, n = 8-10/group


cell cycling status of LT - HSCs after administration of Noggin to HO - 1 + / + or HO - 1 - / - mice .

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cell cycling status of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice.


Media from soluble TREM2 - Fc transfected HEK293T cells incubated with Aβ42 fibrils . Top left panel shows original media samples . Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet . The bottom two panels represent the same samples as the top , but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils . Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet . The bottom two panels represent the same samples as the top , but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1 . All experiments were replicated 3 independent

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

Media from soluble TREM2-Fc transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original media samples. Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times


Media from soluble TREM2 - Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti - Human IgG Fc . Left panels show original media samples . Right panels show the purified soluble TREM2 - Fc ( bottom ) and the presence of APOE3 with the purified soluble TREM2 - Fc ( top ) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1 . All experiments were replicated 3 independent

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

Media from soluble TREM2-Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times


Media from soluble TREM - Fc family member transfected HEK293T cells incubated with Aβ42 fibrils . Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet . Bottom panel shows original media and Aβ42 pellet probed with

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

Media from soluble TREM-Fc family member transfected HEK293T cells incubated with Aβ42 fibrils. Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet. Bottom panel shows original media and Aβ42 pellet probed with 6E10


Media from soluble TREM - Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti - Human IgG Fc . Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3 . Bottom panel shows original media and purified soluble TREM - Fc family members probed with anti -

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

Media from soluble TREM-Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5


Schematic representation of Aβ42 oligomers ( right panel ) and APOE4 ( left panel ) binding with soluble TREM2 - Fc at various concentrations . Curves were fit by setting buffer control ( no ligand ) to y =

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

Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y=0


BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2 - Fc pre incubated with Aβ42 oligomers . Schematic representations of two sequential associations of ligand on soluble TREM2 - Fc are represented by I and II . Soluble TREM2 - Fc loaded on a sensor is first incubated with APOE3 ( association I ) followed by a second incubation with Aβ42 oligomers ( association II ) or the reverse - incubated with Aβ42 oligomers ( association I ) followed by a second incubation with APOE3 ( association II ) . All experiments were replicated 3 independent times

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BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2-Fc pre incubated with Aβ42 oligomers. Schematic representations of two sequential associations of ligand on soluble TREM2-Fc are represented by I and II. Soluble TREM2-Fc loaded on a sensor is first incubated with APOE3 (association I) followed by a second incubation with Aβ42 oligomers (association II) or the reverse- incubated with Aβ42 oligomers (association I) followed by a second incubation with APOE3 (association II). All experiments were replicated 3 independent times


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2 - Fc shows a dose dependent signal . Results were expressed as the OD450 averaged ± standard error ( n = 3 ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

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ELISA plates were coated with purified soluble TREM2-Fc, and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2-Fc shows a dose dependent signal. Results were expressed as the OD450 averaged ± standard error (n =3; * p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM - Fc family members . Results were expressed as the OD450 averaged ± standard error ( n = 4 , * p < 0 . 05 , * * * * p < 0 . 0001 , ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

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ELISA plates were coated with purified soluble TREM2-Fc, and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM-Fc family members. Results were expressed as the OD450 averaged ± standard error (n = 4, * p < 0.05, **** p<0.0001, ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


2B4 NFAT - GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours . GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage ± GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with mock , * * * * p < 0 . 0001 compared with mock , # p < 0 . 05 compared with TREM2 , # # # # p < 0 . 0001 compared with TREM2 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S3 for p value )

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2B4 NFAT-GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours. GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage ± GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with mock, **** p< 0.0001 compared with mock, # p < 0.05 compared with TREM2, #### p < 0.0001 compared with TREM2, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S3 for p value)


B - 2B4 NFAT - GFP reporter cells transduced with TREM2 or variants ( R47H , R62H and T96K ) were incubated with 10 µM Aβ42 oligomers for 12 hours . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with TREM2 , * * p < 0 . 05 compared with TREM2 , * * * * p < 0 . 0001 compared with TREM2 , # # p < 0 . 01 compared with TREM2 T96K , # # # # p < 0 . 0001 compared with TREM2 T96K , ANOVA , Dunnett ' s multiple comparisons test , see Appendix table S3 for p value

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-2B4 NFAT-GFP reporter cells transduced with TREM2 or variants (R47H, R62H and T96K) were incubated with 10 µM Aβ42 oligomers for 12 hours. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with TREM2, ** p < 0.05 compared with TREM2, **** p< 0.0001 compared with TREM2, ## p < 0.01 compared with TREM2 T96K, #### p < 0.0001 compared with TREM2 T96K, ANOVA, Dunnett's multiple comparisons test, see Appendix table S3 for p value


HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA . Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant . Results were averaged ± standard error ( n = 9 , # p < 0 . 05 compared with TREM2 , # # p < 0 . 01 compared with TREM2 , * * * p < 0 . 001 compared with mock , * * * * p < 0 . 0001 compared with mock , ANOVA , Tukey ' s Multiple Comparison Test , see Appendix table S3 for p value )

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HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA. Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant. Results were averaged ± standard error (n = 9, # p < 0.05 compared with TREM2, ## p< 0.01 compared with TREM2, *** p< 0.001 compared with mock, **** p <0.0001 compared with mock, ANOVA, Tukey's Multiple Comparison Test, see Appendix table S3 for p value)


HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours . Conditioned media and cell lysate were analyzed by Western blot for TREM2 ( RIPA lysate ) and soluble TREM2 ( media ) detection . Experiment was replicated 3 independent

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HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours. Conditioned media and cell lysate were analyzed by Western blot for TREM2 (RIPA lysate) and soluble TREM2 (media) detection. Experiment was replicated 3 independent times


HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification . Bottom panel in D shows the presence of TREM2 in the purified product . Endogenous APP and β - tubulin were used as controls for the purification ( middle and upper panels ) . Experiment was replicated 3 independent

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HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification. Bottom panel in D shows the presence of TREM2 in the purified product. Endogenous APP and β-tubulin were used as controls for the purification (middle and upper panels). Experiment was replicated 3 independent time


( a ) Western blot analysis of myosin - VI - depleted HeLa cells untreated or treated with 100 nM bafilomycin A1 . ( b ) Quantification of western blot LC3 - II intensity ( ± s . d . ; n = 3 ) . * * P0 . 01 , * * * P0 .

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(a) Western blot analysis of myosin-VI-depleted HeLa cells untreated or treated with 100 nM bafilomycin A1. (b) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01, ***P0.001


( c ) Confocal immunofluorescence microscopy of LC3 punctae ( green ) in HeLa cells following knockdown of myosin VI . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images ( d ) Quantification of LC3 - and p62 - positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell ( ± s . d . ; n = 3 ) from > 1 , 500 cells per experiment .

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(c) Confocal immunofluorescence microscopy of LC3 punctae (green) in HeLa cells following knockdown of myosin VI. Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images (d) Quantification of LC3- and p62-positive punctae was performed and the results are represented as the average punctae fluorescence intensity per cell (±s.d.; n = 3) from >1,500 cells per experiment.


( e ) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA - resistant GFP - myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132 .

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(e) Western blot analysis of parental HeLa cells or HeLa cells stably expressing siRNA-resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132.


( f ) Western blot analysis of MEFs cultured from wild - type and Snell ' s waltzer ( SV ) mice treated with 1 μM MG132 or 100 nM bafilomycin A1 .

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(f) Western blot analysis of MEFs cultured from wild-type and Snell's waltzer (SV) mice treated with 1 μM MG132 or 100 nM bafilomycin A1.


( g ) Quantification of western blot LC3 - II intensity ( ± s . d . ; n = 3 ) . * * P0 . 01 . ( h ) To evaluate the effects on autophagosome biogenesis , the results are shown as the fold increase in normalized LC3 - II intensity with bafilomycin A1 compared with the untreated control . ( ± s . d . ; n = 3 ) .

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(g) Quantification of western blot LC3-II intensity (±s.d.; n = 3). **P0.01. (h) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control. (±s.d.; n = 3).


( i ) Western blot analysis of cortical neurons from wild - type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1 . ( j ) Quantification of western blot LC3 - II intensity was performed ( n = 2 ) . ( k ) To evaluate the effects on autophagosome biogenesis , the results are shown as the fold increase in normalized LC3 - II intensity with bafilomycin A1 compared with the untreated control ( n = 2 ) .

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(i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM bafilomycin A1. (j) Quantification of western blot LC3-II intensity was performed (n = 2). (k) To evaluate the effects on autophagosome biogenesis, the results are shown as the fold increase in normalized LC3-II intensity with bafilomycin A1 compared with the untreated control (n = 2).


( l ) Western blot ( top ) and confocal immunofluorescence microscopy ( bottom ) of mock - or myosin - VI - siRNA - treated HeLa cells stably expressing the RFP - GFP - LC3 reporter . Hoechst labels the nuclei ( blue ) . ( m ) Quantitative data of RFP and GFP signal overlap from confocal images of mock - or myosin - VI - siRNA - treated HeLa cells expressing RFP - GFP - LC3 . The data are represented as the Pearson ' s coefficient of the RFP and GFP signal correlation from > 100cells per experiment ( ± s . d . ; n = 3 ) .

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(l) Western blot (top) and confocal immunofluorescence microscopy (bottom) of mock- or myosin-VI-siRNA-treated HeLa cells stably expressing the RFP-GFP-LC3 reporter. Hoechst labels the nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock- or myosin-VI-siRNA-treated HeLa cells expressing RFP-GFP-LC3. The data are represented as the Pearson's coefficient of the RFP and GFP signal correlation from >100cells per experiment (±s.d.; n = 3).


n ) Confocal immunofluorescence microscopy of myosin - VI - depleted RPE cells stably expressing GFP - LC3 immunostained against GFP ( green ) and cathepsin D ( red ) . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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n) Confocal immunofluorescence microscopy of myosin-VI-depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h . Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment ( zero time point ) and 8 h post - washout of inhibitor . Immunolabelling for p62 was performed to visualize aggregates ( red ) and Hoechst was used to identify nuclei ( blue ) .

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(a) RPE cells transiently transfected with siRNA against myosin VI and Atg5 were treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy following 16 h MG132 treatment (zero time point) and 8 h post-washout of inhibitor. Immunolabelling for p62 was performed to visualize aggregates (red) and Hoechst was used to identify nuclei (blue).


( b ) Top , western blot analysis confirming siRNA depletion . Bottom , quantification of immunofluorescent p62 - positive aggregates was evaluated using an automated Cellomics VTi microscope . The results were calculated as the average p62 fluorescence intensity at 8 h post - MG132 washout normalized to the zero time point and are represented as the percentage of the control ( ± s . d . ; n = 3 ) . * P0 . 05 , * * * P0 . 001 . See Methods and Supplementary Table S1 for details of the SMARTpool ( SP ) and single ( 07 ) oligonucleotides .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top, western blot analysis confirming siRNA depletion. Bottom, quantification of immunofluorescent p62-positive aggregates was evaluated using an automated Cellomics VTi microscope. The results were calculated as the average p62 fluorescence intensity at 8 h post-MG132 washout normalized to the zero time point and are represented as the percentage of the control (±s.d.; n = 3). *P0.05, ***P0.001. See Methods and Supplementary Table S1 for details of the SMARTpool (SP) and single (07) oligonucleotides.


( c ) Parental HeLa cells or HeLa cells stable expressing siRNA - resistant GFP - myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h . Cells were processed for immunofluorescence microscopy at 16 h post - MG132 treatment ( t = 0 ) or allowed to recover following washout of MG132 for 8 h ( t = 8 ) . Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity . The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout ( t = 8 ; ± s . d . ; n = 3 ) .

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(c) Parental HeLa cells or HeLa cells stable expressing siRNA-resistant GFP-myosin VI were transiently transfected with a single siRNA oligonucleotide targeting myosin VI and were subsequently treated with 1 μM MG132 for 16 h. Cells were processed for immunofluorescence microscopy at 16 h post-MG132 treatment (t = 0) or allowed to recover following washout of MG132 for 8 h (t = 8). Cells were processed for quantification with the automated Cellomics VTi microscope to evaluate the p62 fluorescence intensity. The results represent the fold increase in p62 fluorescence intensity of myosin VI siRNA compared with mock control cells following recovery from MG132 washout (t = 8; ±s.d.; n = 3).


( d ) HeLa cells with stable expression of HttQ72 - GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy . Immunolabelling was performed for GFP ( green ) and p62 ( red ) . Nuclei are labelled with Hoechst ( blue ) . ( e ) Quantification of HttQ72 - GFP aggregates was performed on myosin - VI - siRNA - transfected HeLa cells . The results were calculated as the percentage of GFP - expressing cells with greater than 15 GFP - positive spots per cell . The results represent the mean ( ± s . d . ) from n = 3independent experiments , * * * P0 . 001 . The insets show higher magnifications of the areas outlined in the main images . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(d) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with siRNA against myosin VI followed by saponin extraction and processing for immunofluorescence microscopy. Immunolabelling was performed for GFP (green) and p62 (red). Nuclei are labelled with Hoechst (blue). (e) Quantification of HttQ72-GFP aggregates was performed on myosin-VI-siRNA-transfected HeLa cells. The results were calculated as the percentage of GFP-expressing cells with greater than 15 GFP-positive spots per cell. The results represent the mean (±s.d.) from n = 3independent experiments, ***P0.001. The insets show higher magnifications of the areas outlined in the main images. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) RPE cells transiently co - transfected with GFP - myosin VI and cherry - LC3 and either left untreated ( top row ) or treated with 250 nM Torin1 for 2 h ( bottom row ) were processed for immunofluorescence microscopy . The arrows highlight areas of co - localization and the insets show higher magnifications of the areas outlined in the main images .

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

(a) RPE cells transiently co-transfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 h (bottom row) were processed for immunofluorescence microscopy. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.


( b ) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co - localization . The arrows highlight areas of co - localization and the insets show higher magnifications of the areas outlined in the main images .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RPE cells treated with 100 nM bafilomycin A1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 co-localization. The arrows highlight areas of co-localization and the insets show higher magnifications of the areas outlined in the main images.


( c ) Image stills acquired from spinning - disc time - lapse video microscopy of RPE cells transiently co - transfected with cherry - LC3 and GFP - myosin VI . The outlined regions highlight areas of interest within the cell that are shown to the right ( i - iii ) as video image stills captured at 1 - min intervals . The arrowheads highlight myosin - VI - positive vesicles ( green ) coming into contact and co - localizing with LC3 - positive vesicles ( red ) .

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(c) Image stills acquired from spinning-disc time-lapse video microscopy of RPE cells transiently co-transfected with cherry-LC3 and GFP-myosin VI. The outlined regions highlight areas of interest within the cell that are shown to the right (i-iii) as video image stills captured at 1-min intervals. The arrowheads highlight myosin-VI-positive vesicles (green) coming into contact and co-localizing with LC3-positive vesicles (red).


( d ) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry - LC3 following transient transfection with GFP - myosin VI . Immunostaining for GFP ( green ) and cherry ( red ) was performed and actin was visualized with phalloidin ( white ) . Nuclei were labelled with Hoechst ( blue ) . The arrowheads indicate areas of actin - rich myosin - VI / LC3 - positive autophagosomes . Scale bars , 20 μm ( a , b , d ) ; 10 μm ( c , whole - cell image ) ; 2 μm ( c , cropped images ) .

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(d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualized with phalloidin (white). Nuclei were labelled with Hoechst (blue). The arrowheads indicate areas of actin-rich myosin-VI/LC3-positive autophagosomes. Scale bars, 20 μm (a,b,d); 10 μm (c, whole-cell image); 2 μm (c, cropped images).


( b ) RPE cells with stable expression of cherry - LC3 were transiently transfected with the GFP - myosin VI cargo - binding tail domain containing various mutations in the protein - interaction ( ΔWWY and ΔRRL ) and ubiquitin - binding motifs ( A1013G ) , followed by treatment with 250 nM Torin1 for 3 h to induce autophagy . Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction . The arrowheads indicate areas of co - localization . Scale bar , 20 μm . ( c ) A Pearson ' s coefficient was calculated on the basis of the degree of co - localization between the different GFP - myosin VI mutant tails and cherry - LC3 from confocal immunofluorescence micrographs . The graph represents data from more than 20 transfected cells from n = 2 independent experiments .

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

(b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein-interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 h to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin extraction. The arrowheads indicate areas of co-localization. Scale bar, 20 μm. (c) A Pearson's coefficient was calculated on the basis of the degree of co-localization between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence micrographs. The graph represents data from more than 20 transfected cells from n = 2 independent experiments.


( a ) Left , immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h . Immunolabelling was performed against the indicated endogenous proteins optineurin , T6BP and NDP52 ( red ) , and LC3 ( green ) . The insets show higher magnifications of the areas outlined in the main images . Right , from the confocal images , a Pearson ' s coefficient was calculated to estimate the degree of co - localization of the different autophagy adaptors with LC3 . As a negative control , a Pearson ' s coefficient was calculated after rotation of one colour image by 90 ° . The results represent > 20 cells from n = 2 independent experiments .

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(a) Left, immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM bafilomycin A1 for 2 h. Immunolabelling was performed against the indicated endogenous proteins optineurin, T6BP and NDP52 (red), and LC3 (green). The insets show higher magnifications of the areas outlined in the main images. Right, from the confocal images, a Pearson's coefficient was calculated to estimate the degree of co-localization of the different autophagy adaptors with LC3. As a negative control, a Pearson's coefficient was calculated after rotation of one colour image by 90°. The results represent >20 cells from n = 2 independent experiments.


( b ) Left , stable cherry - LC3 - expressing RPE cells were transiently transfected with GFP - myosin VI , treated with Torin1 for 3 h to induce autophagy , and subsequently processed for confocal immunofluorescence microscopy . Areas of co - localization are white in the merged three - colour images and the insets show higher magnifications of the areas outlined in the main images . Right , a Pearson ' s coefficient was calculated for the degree of co - localization between the three colours . As a negative control , a Pearson ' s coefficient was calculated after rotating one colour image by 90 ° . Results represent > 10 cells from n = 2 independent experiments .

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(b) Left, stable cherry-LC3-expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 h to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of co-localization are white in the merged three-colour images and the insets show higher magnifications of the areas outlined in the main images. Right, a Pearson's coefficient was calculated for the degree of co-localization between the three colours. As a negative control, a Pearson's coefficient was calculated after rotating one colour image by 90°. Results represent >10 cells from n = 2 independent experiments.


( c ) HeLa cells were either mock transfected or co - transfected with siRNA targeted against TNO . Cells were either left untreated or treated with100 nM bafilomycin A1 ( BfnA1 ) for 4 h . Western blot analysis was performed against the indicated proteins . ( d ) Quantification of LC3 - II intensity ( ± s . d . ) was performed from western blot data of TNO - siRNA treated HeLa cells . The results represent n = 3independent experiments . * * P0 . 01 , * * * P0 .

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(c) HeLa cells were either mock transfected or co-transfected with siRNA targeted against TNO. Cells were either left untreated or treated with100 nM bafilomycin A1 (BfnA1) for 4 h. Western blot analysis was performed against the indicated proteins. (d) Quantification of LC3-II intensity (±s.d.) was performed from western blot data of TNO-siRNA treated HeLa cells. The results represent n = 3independent experiments. **P0.01, ***P0.001


( e ) Immunofluorescence microscopy of RFP - GFP - LC3 - expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin . The results are calculated from > 100 cells from n = 2 independent experiments represented as the Pearson ' s coefficient of GFP / RFP signal overlap . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(e) Immunofluorescence microscopy of RFP-GFP-LC3-expressing HeLa cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. The results are calculated from >100 cells from n = 2 independent experiments represented as the Pearson's coefficient of GFP/RFP signal overlap. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Myosin VI and Tom1 / Tom1L2 co - immunoprecipitate from LNCAP cell lysates . Immunoprecipitation of myosin VI with three separate affinity - purified rabbit polyclonal antibodies ( B4 , B7 , 2401 ) was performed alongside an IgG control immunoprecipitation . The immunoprecipitates were analysed by western blotting with antibodies against Tom1 / Tom1L2 .

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(a) Myosin VI and Tom1/Tom1L2 co-immunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity-purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by western blotting with antibodies against Tom1/Tom1L2.


( b ) Binding of myosin VI to Tom1 requires the WWY motif . Bottom , the mammalian two - hybrid assay was used to test binding of full - length Tom1 and Tom1L2 or various truncated versions of Tom1 , annotated by amino acid numbers , against wild - type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail . The graph shows the mean values from n = 2 independent experiments . Top , Tom1 domain structure and truncation mutants . VHS , Vps - 27 , Hrs and STAM ; GAT , GGA and Tom1 .

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(b) Binding of myosin VI to Tom1 requires the WWY motif. Bottom, the mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild-type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n = 2 independent experiments. Top, Tom1 domain structure and truncation mutants. VHS, Vps-27, Hrs and STAM; GAT, GGA and Tom1.


( c ) RPE cells stably expressing GFP - myosin VI were processed for immunofluorescence microscopy to evaluate GFP - myosin VI and endogenous Tom1 / Tom1L2 or Rab5 co - localization . The insets show higher magnifications of the areas outlined in the main panels . The arrows indicate areas of co - localization .

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(c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI and endogenous Tom1/Tom1L2 or Rab5 co-localization. The insets show higher magnifications of the areas outlined in the main panels. The arrows indicate areas of co-localization.


( d ) RPE cells with stable expression of GFP - myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis ( left ) and immunofluorescence microscopy ( middle ) to evaluate GFP - myosin VI tail localization . Cells were either fixed directly or saponin - extracted before fixation . Quantification ( right ) was performed on RPE cells stably expressing GFP - myosin VI tail and transfected with mock , Tom1 or TNO siRNA . Cells were processed for immunocytochemistry , immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP - myosin VI tail punctae per cell using an automated Cellomics VTi microscope . More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP - myosin VI tail punctae per cell ( ± s . d . ) . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by western blot analysis (left) and immunofluorescence microscopy (middle) to evaluate GFP-myosin VI tail localization. Cells were either fixed directly or saponin-extracted before fixation. Quantification (right) was performed on RPE cells stably expressing GFP-myosin VI tail and transfected with mock, Tom1 or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labelled with Hoechst followed by quantification of GFP-myosin VI tail punctae per cell using an automated Cellomics VTi microscope. More than 600 cells per group from n = 3 independent experiments were analysed and are represented as the mean number of GFP-myosin VI tail punctae per cell (±s.d.). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h . Western blot analysis was performed on whole - cell lysates against the indicated proteins . ( b ) Quantification of LC3 - II intensity of western blots was performed by an infrared imaging system . ( ± s . d . ; n = 3 ) . * P0 . 05 , * * P0 . 01 .

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(a) HeLa cells transiently transfected with siRNA against Tom1 were left untreated or treated with 100 nM bafilomycin A1 for 4 h. Western blot analysis was performed on whole-cell lysates against the indicated proteins. (b) Quantification of LC3-II intensity of western blots was performed by an infrared imaging system. (±s.d.; n = 3). *P0.05, **P0.01.


( c , left ) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation ( green ) . The insets show higher magnifications of the areas outlined in the main images . Nuclei are labelled with Hoechst ( blue ) . ( c , right , d ) Quantification of LC3 - positive punctae ( c , right ) and p62 - positive punctae ( d ) was performed in a 96 - well format on an Arrayscan VTi HCS microscope . Cells were identified by Hoechst staining of nuclei ( blue ) . The results are represented as the average punctae fluorescence intensity per cell ( ± s . d . ) from n = 3 independent experiments each performed in triplicate wells , each with > 500 cells .

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(c, left) Confocal immunofluorescence microscopy was performed on HeLa cells transfected with Tom1 siRNA to evaluate LC3 punctae formation (green). The insets show higher magnifications of the areas outlined in the main images. Nuclei are labelled with Hoechst (blue). (c,right,d) Quantification of LC3-positive punctae (c, right) and p62-positive punctae (d) was performed in a 96-well format on an Arrayscan VTi HCS microscope. Cells were identified by Hoechst staining of nuclei (blue). The results are represented as the average punctae fluorescence intensity per cell (±s.d.) from n = 3 independent experiments each performed in triplicate wells, each with >500 cells.


( e ) HeLa cells with stable expression of the RFP - GFP - LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown ( left ) . Confocal immunofluorescence microscopy ( middle ) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin ( right ) . The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson ' s coefficient of GFP / RFP overlap ( ± s . d . ) . A higher Pearson ' s coefficient of GFP / RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes .

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(e) HeLa cells with stable expression of the RFP-GFP-LC3 reporter construct were subjected to mock or Tom1 siRNA mediated knockdown (left). Confocal immunofluorescence microscopy (middle) was performed and images were subsequently quantified for the correlation between the GFP and RFP signals using the ImageJ JACoP plugin (right). The results are for at least 100 individual cells from n = 3 independent experiments and are represented as the Pearson's coefficient of GFP/RFP overlap (±s.d.). A higher Pearson's coefficient of GFP/RFP signal overlap represents a greater number of autophagosomes when compared with autolysosomes.


( f ) Confocal immunofluorescence microscopy of Tom1 - depleted RPE cells stably expressing GFP - LC3 immunostained against GFP ( green ) and cathepsin D ( red ) . Hoechst labels the nuclei ( blue ) . The insets show higher magnifications of the areas outlined in the main images .

(f) Confocal immunofluorescence microscopy of Tom1-depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and cathepsin D (red). Hoechst labels the nuclei (blue). The insets show higher magnifications of the areas outlined in the main images.


( g ) HeLa cells with stable expression of HttQ72 - GFP were transiently transfected with Tom1 siRNA ( top right ) , saponin - extracted , and processed for immunofluorescence microscopy ( left ) to quantify HttQ72 - GFP punctae ( bottom right ) . Immunolabelling against GFP ( green ) and p62 ( red ) was performed . Nuclei ( blue ) were labelled with Hoechst . The results are represented as the number of GFP - expressing cells with greater than 15 GFP - positive spots per cell ( ± s . d . ; n = 3 ) . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(g) HeLa cells with stable expression of HttQ72-GFP were transiently transfected with Tom1 siRNA (top right), saponin-extracted, and processed for immunofluorescence microscopy (left) to quantify HttQ72-GFP punctae (bottom right). Immunolabelling against GFP (green) and p62 (red) was performed. Nuclei (blue) were labelled with Hoechst. The results are represented as the number of GFP-expressing cells with greater than 15 GFP-positive spots per cell (±s.d.; n = 3). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Left , RPE cells with stable expression of GFP - LC3 following mock - or myosin - VI - siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1 / Tom1L2 ( red ) . Nuclei ( blue ) were labelled with Hoechst . The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co - localization and arrowheads highlight adjacent localization of Tom1 relative to LC3 . Right , quantification was performed on > 75 cells for the relative position ( adjacent or complete overlap ) of Tom1 - positive vesicles in relation to LC3 - positive vesicles . The results are represented as the average number of Tom1 - positive vesicles per cell categorized by their relative position to LC3 ( ± s . d . ; n = 3 ) .

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(a) Left, RPE cells with stable expression of GFP-LC3 following mock- or myosin-VI-siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labelled with Hoechst. The insets show higher magnifications of the areas outlined in the main panels and the arrows highlight areas of complete overlapping co-localization and arrowheads highlight adjacent localization of Tom1 relative to LC3. Right, quantification was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. The results are represented as the average number of Tom1-positive vesicles per cell categorized by their relative position to LC3 (±s.d.; n = 3).


( b ) GFP - LC3 - expressing RPE cells were depleted of myosin VI by siRNA . Cells were pulse labelled with Texas - red - dextran ( red ) for 4 h , before chase into fresh medium containing 1 μM MG132 for 2 h . Cells were processed for immunofluorescence microscopy and immunostained for GFP ( green ) . Nuclei were labelled with Hoechst ( blue ) . The insets represent higher magnifications of the areas outlined in the main panels . ( c ) Mander ' s overlap coefficients for the degree of LC3 signal co - localizing with dextran were calculated using Volocity software . The results represent > 20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot . The box represents the median , 25th and 75th percentiles and whiskers represent the maximum and minimum .

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(b) GFP-LC3-expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labelled with Texas-red-dextran (red) for 4 h, before chase into fresh medium containing 1 μM MG132 for 2 h. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labelled with Hoechst (blue). The insets represent higher magnifications of the areas outlined in the main panels. (c) Mander's overlap coefficients for the degree of LC3 signal co-localizing with dextran were calculated using Volocity software. The results represent >20 cells from n = 3 independent experiments and are illustrated as a box and whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum.


( d ) RPE cells stably expressing GFP - LC3 were depleted of myosin VI and Tom1 by siRNA . Cells were pulse - labelled with Texas - red ( TR ) - dextran for 16 h followed by a chase period of 4 h . Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP - LC3 and 5 nm gold particles against Texas - red - dextran . Scale bars , 20 μm ( a , b ) ; 200 nm ( d ) .

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(d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-red (TR)-dextran for 16 h followed by a chase period of 4 h. Cells were processed for immunoelectron microscopy and labelled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas-red-dextran. Scale bars, 20 μm (a,b); 200 nm (d).


B WB showing depletion of PERK in Perk KO astrocytes and neurons .

B WB showing depletion of PERK in Perk KO astrocytes and neurons.


D - I eIF2α phosphorylation in WT and Perk KO astrocytes ( N = 3 ) and neurons ( N = 6 ) ± 20 - 24 hours of TM - induced ER stress . Representative WB ( D , G ) , quantification of absolute phosphorylated eIF2α level ( p - eIF2α / eIF2α ) ( E , H ) and the ER stress - induced p - eIf2α response ( F , I ) . Data information : ( E , H , Data are normalised to untreated WT . ( F , I Data are related to untreated cells of the same genotype . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( E , H , Students t - test ( F , I )

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

D-I eIF2α phosphorylation in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress. Representative WB (D, G), quantification of absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (E, H) and the ER stress-induced p-eIf2α response (F, I). Data information: (E, H, Data are normalised to untreated WT. (F, I Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, H, Students t-test (F, I)


J - O Protein synthesis in WT and Perk KO astrocytes ( N = 5 ) and neurons ( N = 5 ) ± 20 - 24 hours of TM - induced ER stress . Representative images obtained by high - content microscopy showing puromycinilated proteins detected by immunofluorescence ( J , M ) , quantification of puromycin intensity as measure for de novo protein synthesis ( K , N ) and the ER stress - induced protein synthesis response ( L , O ) . Astrocytic soma ( GFAP , white ) , dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized proteins ( Puromycin , green ) . Scale bar : 25 μm . Data information : K , N ) Data are normalised to untreated WT . , L , O ) Data are related to untreated cells of the same genotype . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test K , N ) ; Nested t - test ( L , O ) .

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

J-O Protein synthesis in WT and Perk KO astrocytes (N=5) and neurons (N=5) ± 20-24 hours of TM-induced ER stress. Representative images obtained by high-content microscopy showing puromycinilated proteins detected by immunofluorescence (J, M), quantification of puromycin intensity as measure for de novo protein synthesis (K, N) and the ER stress-induced protein synthesis response (L, O). Astrocytic soma (GFAP, white), dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green). Scale bar: 25 μm. Data information: K, N) Data are normalised to untreated WT. , L, O) Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test K , N); Nested t-test (L, O).


A - F ATF4 expression in WT and Perk KO astrocytes ( N = 3 ) and neurons ( N = 9 ) ± 20 - 24 hours of TM - induced ER stress . ( A , D ) Representative confocal images of ATF4 immunofluorescence . Astrocytic soma ( GFAP , cyan ) , dendrites ( MAP2 , cyan ) , nuclei ( ( Δ ) Cre , green ) , ATF4 ( magenta ) . Scale bar : 10 μm . ( B , E ) Quantification of absolute nuclear ATF4 intensity . Data are normalised to untreated WT . ( C , F ) Quantification of the ER stress - induced ATF4 response . Data are related to untreated cells of the same genotype . Baseline level ( without ER stress ) is depicted by a dashed line . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant p - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , E ) ; Nested t - test ( C , F ) .

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

A-F ATF4 expression in WT and Perk KO astrocytes (N=3) and neurons (N=9) ± 20-24 hours of TM-induced ER stress. (A, D) Representative confocal images of ATF4 immunofluorescence. Astrocytic soma (GFAP, cyan), dendrites (MAP2, cyan), nuclei ((Δ)Cre, green), ATF4 (magenta). Scale bar: 10 μm. (B, E) Quantification of absolute nuclear ATF4 intensity. Data are normalised to untreated WT. (C, F) Quantification of the ER stress-induced ATF4 response. Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F).


G - J ( G , I ) Venn diagram showing the number of significantly upregulated ATF4 target genes in WT ( grey and blue ) and Perk KO ( green and blue ) astrocytes ( G , N = 3 ) and neurons ( I , N = 3 ) upon 20 - 24 hours of TM - induced ER stress determined by mRNA - seq . Percentage : fraction of upregulated ATF4 target genes in WT cells that are also upregulated in Perk KO cells . ( H , N = 3 ; J , N = 3 ) Log2 fold change of ATF4 target genes that are significantly upregulated in both WT and Perk KO cells ( blue in Venn diagram ) . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant p - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant .

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

G-J (G, I) Venn diagram showing the number of significantly upregulated ATF4 target genes in WT (grey and blue) and Perk KO (green and blue) astrocytes (G, N=3) and neurons (I, N=3) upon 20-24 hours of TM-induced ER stress determined by mRNA-seq. Percentage: fraction of upregulated ATF4 target genes in WT cells that are also upregulated in Perk KO cells. (H, N=3; J, N=3) Log2 fold change of ATF4 target genes that are significantly upregulated in both WT and Perk KO cells (blue in Venn diagram). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant.


Protein synthesis and ATF4 expression in mouse ( A - F ) astrocytes ( A - C and neurons ( D - F ± 20 - 24 hours of TM - induced ER stress ± PERK - i ( Pi ) . ( A , D , Representative images obtained by high - content microscopy . Astrocytic soma ( GFAP dendrites ( MAP2 , white ) , nuclei ( ΔCre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . ( B , N = 5 ; E , N = 5 ; Quantification of puromycin intensity as measure for de novo protein synthesis . ( C , N = 5 ; F , N = 7 ; Quantification of absolute nuclear ATF4 intensity . Data information : Data are normalised to untreated WT and presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test .

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

Protein synthesis and ATF4 expression in mouse (A-F) astrocytes (A-C and neurons (D-F ± 20-24 hours of TM-induced ER stress ± PERK-i (Pi). (A, D, Representative images obtained by high-content microscopy. Astrocytic soma (GFAP dendrites (MAP2, white), nuclei (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (B, N=5; E, N=5; Quantification of puromycin intensity as measure for de novo protein synthesis. (C, N=5; F, N=7; Quantification of absolute nuclear ATF4 intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.


Protein synthesis and ATF4 expression in human ( G - L ) astrocytes G - I , ) and neurons J - L ) ± 20 - 24 hours of TM - induced ER stress ± PERK - i ( Pi ) . G , J ) Representative images obtained by high - content microscopy . Astrocytic soma Phalloidin , white ) , dendrites ( MAP2 , white ) , nuclei ( ΔCre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . ; H , N = 3 ; K , N = 3 ) Quantification of puromycin intensity as measure for de novo protein synthesis . I , N = 4 ; L , N = 3 ) Quantification of absolute nuclear ATF4 intensity . Data information : Data are normalised to untreated WT and presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test .

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

Protein synthesis and ATF4 expression in human (G-L) astrocytes G-I, ) and neurons J-L) ± 20-24 hours of TM-induced ER stress ± PERK-i (Pi). G, J) Representative images obtained by high-content microscopy. Astrocytic soma Phalloidin, white), dendrites (MAP2, white), nuclei (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. ; H, N=3; K, N=3) Quantification of puromycin intensity as measure for de novo protein synthesis. I, N=4; L, N=3) Quantification of absolute nuclear ATF4 intensity. Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test.


D - G ROS accumulation in WT and Perk KO neurons ( N = 3 ) and astrocytes ( N = 3 ) ± 20 - 24 hours of TM - induced ER stress . ( D , F ) Representative images obtained by high - content microscopy . Dendrites ( MAP2 , white ) , astrocytic soma ( Phalloidin , white ) , nuclei ( ( Δ ) Cre , red ) , ROS ( CellROX green , green ) . Scale bar : 25 μm . ( E , G ) Quantification of CellROX green intensity . Data information : ( E , G , Data are normalised to untreated WT . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( E , G ,

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

D-G ROS accumulation in WT and Perk KO neurons (N=3) and astrocytes (N=3) ± 20-24 hours of TM-induced ER stress. (D, F) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), astrocytic soma (Phalloidin, white), nuclei ((Δ)Cre, red), ROS (CellROX green, green). Scale bar: 25 μm. (E, G) Quantification of CellROX green intensity. Data information: (E, G, Data are normalised to untreated WT. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, G,


H mRNA expression of Eif2ak1 ( HRI ) in HRI KD neurons determined by RT - qPCR , normalised to WT transduced with shRNA control ( represented by a dashed line ) ( N = 3 ) . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis One sample t - test with a hypothetical value of 100 (

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

H mRNA expression of Eif2ak1 (HRI) in HRI KD neurons determined by RT-qPCR, normalised to WT transduced with shRNA control (represented by a dashed line) (N=3). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis One sample t-test with a hypothetical value of 100 (H


I Quantification of the increase in nuclear ATF4 intensity in WT neurons ± HRI KD ± 20 hours of sodium arsenite ( Ars ) treatment ( N = 3 ) . J The remaining ATF4 response ( % ) upon HRI KD , related to ATF4 response of WT transduced with shRNA control ( 100 % , represented by a dashed line ) ( N = 3 ) . Calculated from data in ( I ) : [ [ ( KD _ Ars + - KD _ Ars - ) / KD _ Ars - ] / [ ( WT _ Ars + - WT _ Ars - ) / WT _ Ars - ] ] * 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test I , One sample t - test with a hypothetical value of 100 J )

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

I Quantification of the increase in nuclear ATF4 intensity in WT neurons ± HRI KD ± 20 hours of sodium arsenite (Ars) treatment (N=3). J The remaining ATF4 response (%) upon HRI KD, related to ATF4 response of WT transduced with shRNA control (100%, represented by a dashed line) (N=3). Calculated from data in (I): [[(KD_Ars+-KD_Ars-)/KD_Ars-]/[(WT_Ars+-WT_Ars-)/WT_Ars-]]*100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test I, One sample t-test with a hypothetical value of 100 J)


K - M eIF2α phosphorylation in HRI KD WT and Perk KO neurons ( N = 3 ) ± 20 - 24 hours of TM - induced ER stress . Representative WB ( K ) , quantification of the ER stress - induced p - eIf2α response ( L ) and absolute phosphorylated eIF2α level ( p - eIF2α / eIF2α ) ( M ) . Data information : M , Data are normalised to untreated WT . ( L , Data are related to untreated cells of the same genotype and normalised to WT without HRI KD . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test L , Students t - test ( M )

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

K-M eIF2α phosphorylation in HRI KD WT and Perk KO neurons (N=3) ± 20-24 hours of TM-induced ER stress. Representative WB (K), quantification of the ER stress-induced p-eIf2α response (L) and absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (M). Data information: M, Data are normalised to untreated WT. (L, Data are related to untreated cells of the same genotype and normalised to WT without HRI KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test L, Students t-test (M)


N - S Protein synthesis ( N = 3 ) and ATF4 expression ( N = 4 ) in HRI KD WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress . Representative images obtained by ( N ) confocal and ( Q ) high - content microscopy . Dendrites ( MAP2 , white / cyan ) , nuclei ( ( Δ ) Cre , green / red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis response ( O ) and puromycin intensity as measure for de novo protein synthesis ( P ) . Quantification of the ER stress - induced ATF4 response ( R ) and absolute nuclear ATF4 intensity ( S ) . Data information : P , S ) Data are normalised to untreated WT . O , R ) Data are related to untreated cells of the same genotype and normalised to WT without HRI KD . Baseline level ( without ER stress ) is depicted by a dashed line . Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test O , R ) ; Nested t - test ( P , S ) .

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

N-S Protein synthesis (N=3) and ATF4 expression (N=4) in HRI KD WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress. Representative images obtained by (N) confocal and (Q) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, green/red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis response (O) and puromycin intensity as measure for de novo protein synthesis (P). Quantification of the ER stress-induced ATF4 response (R) and absolute nuclear ATF4 intensity (S). Data information: P, S) Data are normalised to untreated WT. O, R) Data are related to untreated cells of the same genotype and normalised to WT without HRI KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, R); Nested t-test (P, S).


A - F Protein synthesis ( A - C , N = 3 ) and ATF4 expression ( D - F , N = 5 ) in WT and Perk KO neurons overexpressing eIF2αWT or phospho - deficient eIF2αS51A ± 20 - 24 hours of TM - induced ER stress . Representative images obtained by ( A ) confocal and ( D ) high - content microscopy . Dendrites ( MAP2 , white / cyan ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis ( B ) and ATF4 response ( E ) . Data are normalised to WT eIF2αWT . Quantification showing the percentage change in the ER stress - induced protein synthesis ( C ) and ATF4 ( F ) response induced by eIF2αS51A compared to eIF2αWT . ( C ) Calculated from data in ( B ) : [ ( eIF2αS51A - eIF2αWT ) / eIF2αWT ] * 100 . ( F ) Calculated from data in ( E ) : [ ( eIF2αS51A - eIF2αWT ) / eIF2αWT ] * - 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , E , Nested t - test ( C , F ) .

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

A-F Protein synthesis (A-C, N=3) and ATF4 expression (D-F, N=5) in WT and Perk KO neurons overexpressing eIF2αWT or phospho-deficient eIF2αS51A ± 20-24 hours of TM-induced ER stress. Representative images obtained by (A) confocal and (D) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (B) and ATF4 response (E). Data are normalised to WT eIF2αWT. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by eIF2αS51A compared to eIF2αWT. (C) Calculated from data in (B): [(eIF2αS51A-eIF2αWT)/eIF2αWT] *100. (F) Calculated from data in (E): [(eIF2αS51A-eIF2αWT)/eIF2αWT]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E, Nested t-test (C, F).


Expression of p - eIF2α sensitive ( 5 ' ATF4 - uORFWT ) and p - eIf2α insensitive ( 5 ' ATF4 - uORFMut ) EYFP translational reporters in WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 3 ) . ( I , K ) Representative confocal images . Dendrites ( MAP2 , cyan ) , nuclei ( ( Δ ) Cre , red ) , endogenous ATF4 ( magenta ) , ATF4 reporter ( green ) . Scale bar : 10 μm . ( J , L ) Quantification of EYFP intensity , normalised to untreated WT . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test J , L )

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

Expression of p-eIF2α sensitive (5'ATF4-uORFWT) and p-eIf2α insensitive (5'ATF4-uORFMut) EYFP translational reporters in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=3). (I, K) Representative confocal images. Dendrites (MAP2, cyan), nuclei ((Δ)Cre, red), endogenous ATF4 (magenta), ATF4 reporter (green). Scale bar: 10 μm. (J, L) Quantification of EYFP intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test J, L)


A - F Protein synthesis and ATF4 expression in ISRIB - treated WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 4 ) . ( A , D ) Representative images obtained by high - content microscopy . Dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis ( B ) and ATF4 ( E ) response . Data are normalised to WT without ISRIB . Baseline level ( without ER stress ) is depicted by dashed line . Quantification showing the percentage change in the ER stress - induced protein synthesis ( C ) and ATF4 ( F ) response induced by ISRIB . ( C ) Calculated from data in ( B ) : [ ( ISRIB + - ISRIB - ) / ISRIB - ] * 100 . ( F ) Calculated from data in ( E ) : [ ( ISRIB + - ISRIB - ) / ISRIB - ] * - 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , E ) ; Nested t - test ( C , F )

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

A-F Protein synthesis and ATF4 expression in ISRIB-treated WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (A, D) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (B) and ATF4 (E) response. Data are normalised to WT without ISRIB. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (C) and ATF4 (F) response induced by ISRIB. (C) Calculated from data in (B): [(ISRIB+-ISRIB-)/ISRIB-]*100. (F) Calculated from data in (E): [(ISRIB+-ISRIB-)/ISRIB-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F)


G - M Protein synthesis and ATF4 expression in 2b5he and 2b5ho astrocytes and neurons ± 20 - 24 hours of TM - induced ER stress in the presence or absence of PERK - i ( Pi ) ( N = 3 ) . ( G , I , K , M ) Representative images obtained by high - content microscopy . Astrocytic soma ( Phalloidin , white ) , dendrites ( MAP2 , white ) , nuclei ( DAPI , red ) , de novo synthesized proteins ( Puromycin , green ) , ATF4 ( magenta ) . Scale bar : 25 μm . ( H , L ) Quantification of puromycin intensity as measure for de novo protein synthesis and ( J , N ) absolute nuclear ATF4 intensity . Data are normalised to untreated WT . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 and ns : not significant . Statistical analysis : Three - way ANOVA with Tukey ' s post hoc test ( H , J , L , N ) .

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

G-M Protein synthesis and ATF4 expression in 2b5he and 2b5ho astrocytes and neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of PERK-i (Pi) (N=3). (G, I, K, M) Representative images obtained by high-content microscopy. Astrocytic soma (Phalloidin, white), dendrites (MAP2, white), nuclei (DAPI, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (H, L) Quantification of puromycin intensity as measure for de novo protein synthesis and (J, N) absolute nuclear ATF4 intensity. Data are normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, *** p<0.001, **** p<0.0001 and ns: not significant. Statistical analysis: Three-way ANOVA with Tukey's post hoc test (H, J, L, N).


A Differentially expressed genes ( DEGs ) involved in oxidative stress related processes comparing TM - treated Perk KO and WT neurons . Blue arrow : Ang .

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

A Differentially expressed genes (DEGs) involved in oxidative stress related processes comparing TM-treated Perk KO and WT neurons. Blue arrow: Ang.


B , C Ang mRNA level in WT and Perk KO neurons and astrocytes ± 20 - 24 hours of TM - induced ER stress , determined by mRNA - seq ( N = 3 ) . FPMK : Fragments Per Kilobase of transcript per Million . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test ( B , C ,

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C Ang mRNA level in WT and Perk KO neurons and astrocytes ± 20-24 hours of TM-induced ER stress, determined by mRNA-seq (N=3). FPMK: Fragments Per Kilobase of transcript per Million. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, C,


D , E Representative WB and quantification of ANG protein level ( N = 3 ) . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test E ,

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

D, E Representative WB and quantification of ANG protein level (N=3). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test E,


F , G tRNA unfolding in WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress ( N = 4 ) . ( F ) Representative images obtained by high - content microscopy of 1 - methyladenosine ( m1A ) immunofluorescence to determine tRNA unfolding . Dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , unfolded tRNA ( m1A , green ) . Scale bar : 25 μm . ( G ) Quantification of m1A intensity , normalised to untreated WT . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test G ,

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

F, G tRNA unfolding in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress (N=4). (F) Representative images obtained by high-content microscopy of 1-methyladenosine (m1A) immunofluorescence to determine tRNA unfolding. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), unfolded tRNA (m1A, green). Scale bar: 25 μm. (G) Quantification of m1A intensity, normalised to untreated WT. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test G,


H - M Abundance of small RNAs in WT and Perk KO astrocytes ( N = 3 ) and neurons ( N = 6 ) ± 20 - 24 hours of TM - induced ER stress , determined by high - resolution automated electrophoresis ( H , K ) Representative gel image . Electropherogram ( I , L ) and zoom ( J , M ) showing quantified fluorescence units ( FU ) of small RNAs , size ranges of tRNAs and tsncRNAs are indicated . Shown is the mean of the biological replicates .

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

H-M Abundance of small RNAs in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress, determined by high-resolution automated electrophoresis (H, K) Representative gel image. Electropherogram (I, L) and zoom (J, M) showing quantified fluorescence units (FU) of small RNAs, size ranges of tRNAs and tsncRNAs are indicated. Shown is the mean of the biological replicates.


N - R Protein synthesis and ATF4 expression in WT and Perk KO neurons ± 20 - 24 hours of TM - induced ER stress in the presence or absence of ANG - I ( N = 3 ) . ( N ) Representative images obtained by high - content microscopy . Dendrites ( MAP2 , white ) , nuclei ( ( Δ ) Cre , red ) , de novo synthesis of proteins ( Puromycin , green ) . Scale bar : 25 μm . Quantification of the ER stress - induced protein synthesis ( O ) and ATF4 response ( Q ) . Data are normalised to WT without ANG - i . Baseline level ( without ER stress ) is depicted by dashed line . Quantification showing the percentage change in the ER stress - induced protein synthesis ( P ) and ATF4 response ( R ) induced by ANG - i . ( P ) Calculated from data in ( O ) : [ ( ANG - i + - ANG - i - ) / ANG - i - ] * 100 . ( R ) Calculated from data in ( Q ) : [ ( ANG - i + - ANG - i - ) / ANG - i - ] * - 100 . Data information : Data are presented as mean ± SEM . N : Biological replicate . Relevant P - values are indicated : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Statistical analysis : Two - way ANOVA with Tukey ' s post hoc test O , Q ) ; Nested t - test ( P , R ) .

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

N-R Protein synthesis and ATF4 expression in WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress in the presence or absence of ANG-I (N=3). (N) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesis of proteins (Puromycin, green). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis (O) and ATF4 response (Q). Data are normalised to WT without ANG-i. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress-induced protein synthesis (P) and ATF4 response (R) induced by ANG-i. (P) Calculated from data in (O): [(ANG-i+-ANG-i-)/ANG-i-]*100. (R) Calculated from data in (Q): [(ANG-i+-ANG-i-)/ANG-i-]*-100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, Q); Nested t-test (P, R).


B - C . Graphs showing the relative weight ( B ; n = 15 WT , 12 ECko ; curve comparison ) and the survival ( C ; n = 15 WT , 11 ECko ; Log - Rank test ) of mice upon tamoxifen treatment . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

B-C. Graphs showing the relative weight (B; n= 15 WT, 12 ECko; curve comparison) and the survival (C; n= 15 WT, 11 ECko; Log-Rank test) of mice upon tamoxifen treatment. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


D . Representative images of the intestine of Casp8WT and Casp8ECko mice , with mild and severe hemorrhages at a late disease stage .

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

D. Representative images of the intestine of Casp8WT and Casp8ECko mice, with mild and severe hemorrhages at a late disease stage.


E - F . Representative images of H & E staining ( E ) and quantification of intestinal pathology ( F ) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice ( enteropathy score ; n = 6 WT , 6 ECko ; two tailed unpaired Student ' s t - test ) . Scale bars : 100µm ; insets 50µm . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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

E-F. Representative images of H&E staining (E) and quantification of intestinal pathology (F) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice (enteropathy score; n= 6 WT, 6 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm; insets 50µm. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


G - H . Quantification of intestinal pathology ( enteropathy score , G ; n = 8 WT , 7 ECko ; two tailed unpaired Student ' s t - test ) and hemorrhages into the mucosa ( H ; n = 4 WT , 5 ECko ; two tailed unpaired Student ' s t - test ) at a late disease stage in Casp8WT and Casp8ECko mice . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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

G-H. Quantification of intestinal pathology (enteropathy score, G; n= 8 WT, 7 ECko; two tailed unpaired Student's t-test) and hemorrhages into the mucosa (H; n= 4 WT, 5 ECko; two tailed unpaired Student's t-test) at a late disease stage in Casp8WT and Casp8ECko mice. Data information: Data shown as mean ± SEM. **P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


I . Overview of H & E stained swiss roll preparations of Casp8WT and Casp8ECko mice . Scale bars : 2mm .

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

I. Overview of H&E stained swiss roll preparations of Casp8WT and Casp8ECko mice. Scale bars: 2mm.


J . Representative images of H & E staining of the small intestine of Casp8WT ( j ' ) and Casp8ECko mice with mild ( j ' ' ) and more severely ( j ' ' ' ) affected areas at a late disease stage ( white arrows point to hemorrhages , asterisk points to focal erosion of the epithelium ) . Scale bars : 100µm .

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

J. Representative images of H&E staining of the small intestine of Casp8WT (j') and Casp8ECko mice with mild (j'') and more severely (j''') affected areas at a late disease stage (white arrows point to hemorrhages, asterisk points to focal erosion of the epithelium). Scale bars: 100µm.


A - B . QPCR analysis of genes associated to immune and bacterial response ( A ) , vessel permeability and EC activation ( B ) in small intestine lysates at a late disease timepoint in Casp8WT and Casp8ECko mice ( n = 4 WT , 3 - 4 ECko , biological replicates , multiple t - test with Holm - Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

A-B. QPCR analysis of genes associated to immune and bacterial response (A), vessel permeability and EC activation (B) in small intestine lysates at a late disease timepoint in Casp8WT and Casp8ECko mice (n= 4 WT, 3-4 ECko, biological replicates, multiple t-test with Holm-Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.