Figure 8B . SH - SY5Y cells co - expressing Q97 - GFP with MIA40 ( middle row ) , ∆ N - MIA40 ( lower row ) or a mock - transfected control ( upper row ) were analyzed by immunocytochemistry and super - resolution microscopy . The magnification shows an enlarged section of the TOM20 staining . TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix , respectively . Bars , 20 µm . Magnifications : 40fold of a 63x objective field . D . SH - SY5Y cells co - expressing Q97 - GFP with MIA40 ( upper row ) or ∆ N - MIA40 ( lower row ) were analyzed by immunocytochemistry and super - resolution microscopy . The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix , respectively . Bars , 20 µm . Magnifications : 40fold of a 63x objective field .

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Figure 8B. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (middle row), ∆N-MIA40 (lower row) or a mock-transfected control (upper row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TOM20 staining. TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.D. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (upper row) or ∆N-MIA40 (lower row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.


Figure 1Immunoblot analysis of presenilin expression and endoproteolysis in cell lysates of untransfected HEK293 / sw cells endogenously expressing presenilin and single cell clones overexpressing PS1 WT or the indicated PS1 mutants . Immunoblot analysis of total Aβ secretion and the production of different Aβ species ( shown in low and high immunoblot exposures ) in conditioned media of cell linesMass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of cell

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Figure 1Immunoblot analysis of presenilin expression and endoproteolysis in cell lysates of untransfected HEK293/sw cells endogenously expressing presenilin and single cell clones overexpressing PS1 WT or the indicated PS1 mutants.Immunoblot analysis of total Aβ secretion and the production of different Aβ species (shown in low and high immunoblot exposures) in conditioned media of cell linesMass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of cell lines


Figure 2Immunoblot analysis of C99 - PS1 NTF or full - length ( FL ) crosslinks ( CL ) of the indicated mutants . Samples that were not UV - irradiated were loaded to control for specificity . Changes in the ratio of crosslink efficiencies at positions T48 and L49 compared to corresponding controls ( n = 4 - 5 biological replicates ) . Changes in the ratio of crosslink efficiencies at positions K54 and V44 compared to corresponding controls ( n = 4 - 5 biological replicates ) .

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Figure 2Immunoblot analysis of C99-PS1 NTF or full-length (FL) crosslinks (CL) of the indicated mutants. Samples that were not UV-irradiated were loaded to control for specificity.Changes in the ratio of crosslink efficiencies at positions T48 and L49 compared to corresponding controls (n = 4-5 biological replicates). Changes in the ratio of crosslink efficiencies at positions K54 and V44 compared to corresponding controls (n = 4-5 biological replicates).


Figure 3ELISA analysis of Aβ species in conditioned medium of HEK293 / sw cells treated with RO7019009 or vehicle ( DMSO ) . Aβ levels are shown relative to DMSO vehicle control ( n = 4 biological replicates ) . Dose response curve of RO7019009 in HEK293 / sw cells measured using the MSD sandwich - immunoassay ( n = 3 biological replicates ) . Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines . Comparison of in vitro potencies for Aβ42 inhibition in HEK293 / sw cells , IC50 = 14 nM ( n = 3 ) ( as in ( C ) ) with those in H4 / sw cells , IC50 = 5 nM ( n = 7 ) and N2a / sw cells , IC50 = 2 nM ( n = 6 ) together with that of Notch cleavage inhibition in a HEK293 - based Notch1 luciferase reporter cell line , IC50 > 10 µM ( n = 2 ) ( all n numbers represent biological replicates ) . Left panel : Immunoblot analysis of total Aβ in conditioned media of HEK293 / sw cells treated with RO7019009 or vehicle ( DMSO ) . Total APPs levels were analyzed to control for normal APP secretion and equal sample loading . Right panel : Quantification of relative Aβ amounts in ( E ) ( n = 2 biological replicates ) . Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293 / sw cells after separation by Tris - Bicine urea SDS PAGE ; synthetic Aβ peptides were loaded as size markers .

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Figure 3ELISA analysis of Aβ species in conditioned medium of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Aβ levels are shown relative to DMSO vehicle control (n = 4 biological replicates).Dose response curve of RO7019009 in HEK293/sw cells measured using the MSD sandwich-immunoassay (n = 3 biological replicates).Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines. Comparison of in vitro potencies for Aβ42 inhibition in HEK293/sw cells, IC50 = 14 nM (n = 3) (as in (C)) with those in H4/sw cells, IC50 = 5 nM (n = 7) and N2a/sw cells, IC50 = 2 nM (n = 6) together with that of Notch cleavage inhibition in a HEK293-based Notch1 luciferase reporter cell line, IC50 > 10 µM (n = 2) (all n numbers represent biological replicates).Left panel: Immunoblot analysis of total Aβ in conditioned media of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Total APPs levels were analyzed to control for normal APP secretion and equal sample loading. Right panel: Quantification of relative Aβ amounts in (E) (n = 2 biological replicates).Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293/sw cells after separation by Tris-Bicine urea SDS PAGE; synthetic Aβ peptides were loaded as size markers.


Figure 4ELISA analysis of Aβ species in conditioned media of HEK293 / sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle ( DMSO ) showing the secretion of Aβ38 ( A ) , Aβ43 ( B ) and Aβ42 ( C ) compared to total Aβ ( Aβ38 + Aβ40 + Aβ42 + Aβ43 ) ( n = 5 biological replicates ) . Note that in some experiments Aβ38 generation was below the detection limit for some mutants . Data are presented as mean ± SD .

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Figure 4ELISA analysis of Aβ species in conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle (DMSO) showing the secretion of Aβ38 (A), Aβ43 (B) and Aβ42 (C) compared to total Aβ (Aβ38 + Aβ40 + Aβ42 + Aβ43) (n = 5 biological replicates). Note that in some experiments Aβ38 generation was below the detection limit for some mutants. Data are presented as mean ± SD.


Figure 5Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293 / sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle ( DMSO ) .

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Figure 5Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle (DMSO).


Figure 6Immunoblot analysis of C99 V44Bpa crosslinking ( CL ) to PS1 WT or NTFs ( circle ) of normally endoproteolysed mutants in the presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in ( A ) . Immunoblot analysis of C99 V44Bpa crosslinking to full - length ( FL ) PS1 ( triangle ) of endoproteolysis - deficient mutants and the corresponding M292D control in the presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in ( C ) .

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Figure 6Immunoblot analysis of C99 V44Bpa crosslinking (CL) to PS1 WT or NTFs (circle) of normally endoproteolysed mutants in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in (A). Immunoblot analysis of C99 V44Bpa crosslinking to full-length (FL) PS1 (triangle) of endoproteolysis-deficient mutants and the corresponding M292D control in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in (C).


Figure 1IES retention PCR ( cropped inverted images ) . Four maternally - controlled IES and four non - maternally controlled IESs are shown . The IES + band represents retained IES ; the IES - band represents an excised IES ; additional bands are likely PCR artefacts . IRS is IES retention Score for the IESs calculated after whole genome sequencing .

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Figure 1IES retention PCR (cropped inverted images). Four maternally-controlled IES and four non-maternally controlled IESs are shown. The IES+ band represents retained IES; the IES- band represents an excised IES; additional bands are likely PCR artefacts. IRS is IES retention Score for the IESs calculated after whole genome sequencing.


Figure 3 ( A ) Schematic drawing of the life cycle stages of Paramecium tetraurelia . MIC and parental MAC are represented in red , representing the DAPI signal , and developing MAC ( dm ) is represented in green until fully developed , representing the GFP signal . ( B ) ISWI1 - tagged C - terminally with GFP localizes in the developing MAC as soon as developing new MACs ( panel Early Development ) become visible and remain there throughout late MAC development ( panel Late Development ) . Red : DAPI , Green : ISWI1 - GFP . Blue arrows identify developing MAC ; ( C ) Western blot analysis using anti - HA antibody after coimmunoprecipitation of ISWI1 - 3XFlagHA fusion protein . Non - transformed cells ( WT ) of the same strain were used as the negative control . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 12 % SDS gel . ( D ) Volcano plot illustrating the distribution of proteins identified in label - free MS in WT Vs ISWI1 - 3XFlagHA . Significantly abundant proteins ( fold change ≥ 4 ) are highlighted in orange . ( E ) Western blot analysis using anti - HA and anti - GFP antibodies after coimmunoprecipitation of Ptiwi01 - 3XFlagHA fusion protein co - transformed with ISWI1 - GFP . Non - transformed cells ( WT ) of the same strain and ISWI1 - GFP fusion protein transformation were used as negative controls . 1 % of the total lysate was loaded as Input and 20 % of co - immunoprecipitated samples were loaded on 10 % SDS gel .

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Figure 3(A) Schematic drawing of the life cycle stages of Paramecium tetraurelia. MIC and parental MAC are represented in red, representing the DAPI signal, and developing MAC (dm) is represented in green until fully developed, representing the GFP signal. (B) ISWI1-tagged C-terminally with GFP localizes in the developing MAC as soon as developing new MACs (panel Early Development) become visible and remain there throughout late MAC development (panel Late Development). Red: DAPI, Green: ISWI1-GFP. Blue arrows identify developing MAC;(C) Western blot analysis using anti-HA antibody after coimmunoprecipitation of ISWI1-3XFlagHA fusion protein. Non-transformed cells (WT) of the same strain were used as the negative control. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 12% SDS gel.(D) Volcano plot illustrating the distribution of proteins identified in label-free MS in WT Vs ISWI1-3XFlagHA. Significantly abundant proteins (fold change ≥ 4) are highlighted in orange.(E) Western blot analysis using anti-HA and anti-GFP antibodies after coimmunoprecipitation of Ptiwi01-3XFlagHA fusion protein co-transformed with ISWI1-GFP. Non-transformed cells (WT) of the same strain and ISWI1-GFP fusion protein transformation were used as negative controls. 1% of the total lysate was loaded as Input and 20% of co-immunoprecipitated samples were loaded on 10%SDS gel.


Figure 1B . Internalization ( cytoplasm ) and recycling ( plasma membrane ) of FGF7 - and FGF10 - stimulated endogenous FGFR2 ( green ) for 0 , 15 , 40 , and 120 min . in T47D . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , cells with receptor recycled to the plasma membrane . Scale bar , 5 μm . C . Red and green pixels overlap fraction representing the co - localization of FGFR2b with the recycling endosomes marker Tf ( above ) and the proportion of green over total pixels representing FGFR2b in the cytoplasm ( below ) upon stimulation with FGF7 ( dark green ) or FGF10 ( burgundy ) for 15 , 40 , or 120 min . . Values represent the median ± SD of at least 3 independent experiments . Representative pictures are shown in B ( cytoplasm ) . * , p value < 0 . 005 ( Student ' s t - test ) . D H . Internalization ( cytoplasm ) and recycling ( plasma membrane ) of FGF10 - stimulated HA - FGFR2b ( green ) transfected in BT549 stimulated for 0 , 40 and 120 min . . Cells were depleted or not of TTP or RCP by a pool of siRNAs . TRITC - Tf is a marker of recycling ( red ) . Nuclei are stained in blue . * , cells with receptor recycled to the plasma membrane . Scale bar , 5 μm .

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Figure 1B. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test). D H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.


Figure 2 ( J ) control or 40 min . FGF10 - or TGFα - stimulated T47D left untreated or depleted of TTP , followed or not by transfection with siRNA resistant Flag - TTP ( Flag - TTP * ) or depleted of RCP followed or not by transfection with siRNA resistant RCP - GFP ( RCP - GFP * ) were immunoblotted with the indicated antibodies . * * , non - specific band ( J ) .

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Figure 2(J) control or 40 min. FGF10- or TGFα-stimulated T47D left untreated or depleted of TTP, followed or not by transfection with siRNA resistant Flag-TTP (Flag-TTP*) or depleted of RCP followed or not by transfection with siRNA resistant RCP-GFP (RCP-GFP*) were immunoblotted with the indicated antibodies. **, non-specific band (J).


Figure 3F , G Lysates from T47D or BT20 treated with DMSO , PD173074 , AG1478 , or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα ( F ) ; T47D or BT20 treated with DMSO , or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα ( G ) were immunoblotted with the indicated antibodies .

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Figure 3F, G Lysates from T47D or BT20 treated with DMSO, PD173074, AG1478, or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα (F); T47D or BT20 treated with DMSO, or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα (G) were immunoblotted with the indicated antibodies.


Figure 4Lysates from T47D stimulated or not with FGF10 , EGF for different time periods or treated with PD173074 ( C ) , MEK162 ( D ) , AG1478 ( E ) before stimulation were immunoblotted with the indicated antibodies . G , H Percentage of EdU incorporation in T47D pre - treated or not with FGF10 for 40 min . and stimulated or not with FGF10 or EGF ( G ) or incubated with MEK162 , PD173074 , or AG1478 before pre - treatment ( H ) . N = 6 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey test ) .

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Figure 4Lysates from T47D stimulated or not with FGF10, EGF for different time periods or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies.G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


Figure 5A . Co - localization of FGFR2 ( red ) , EGFR ( green ) , and the recycling marker Tf ( blue ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 μm . C . Co - localization of EGFR ( green ) , T693 phosphorylated EGFR ( blue ) , and the recycling marker Tf ( red ) in T47D stimulated or not with FGF10 or TGFα for 40 min . Scale bar , 5 μm . E . Co - localization of FGFR2 ( red ) , EGFR ( green ) , and the recycling marker Tf ( blue ) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods . Scale bar , 5 μm .

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Figure 5A. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm. C. Co-localization of EGFR (green), T693 phosphorylated EGFR (blue), and the recycling marker Tf (red) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm.E. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods. Scale bar, 5 μm.


Figure 6E , F Lysates from T47D ( E ) and BT20 ( F ) depleted of EGFR , transfected with wt or T693A , and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies .

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Figure 6E, F Lysates from T47D (E) and BT20 (F) depleted of EGFR, transfected with wt or T693A, and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies.


Figure 7A - D Percentage of EdU incorporation in T47D ( A , C ) or BT20 ( B , D ) depleted of EGFR or not , transfected with wt or T693A , and stimulated or not with FGF10 ( A , B ) or with TGFα ( C , D ) and pre - treated ( C , D ) or not ( A , B ) with FGF10 for 40 min . Values represent the median ± SD of N = 4 . P value = < 0 . 05 * , < 0 . 01 * * , < 0 . 001 * * * ( one - way ANOVA with Tukey test ) .

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Figure 7A-D Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).


Figure 1A , B Temporal expression of Snail genes in the intestinal tract in ( A ) embryonic E12 . 5 to E18 . 5 and ( B ) postnatal P0 to P20 measured by qRT - PCR . Shown are means ± SD . C , E , G , I Immunohistochemical staining for Snai1 / 2 ( Abcam ab85931 ) ( C ) E14 . 5midgut , ( E ) E14 . 5hind gut , ( G ) postnatal day 14 SI , ( I ) postnatal day 14 colon . Snai1immunohistochemistry peptide competition controls are shown as inserts ( C ' , E ' , G ' , I ' ) . Scale bars : 20 mm . D , F , H , J , K X ‐ gal staining for β ‐ galactosidase activity in tissue from Snai2LacZ mice stained in whole mount , sectioned and counterstained with nuclear fast red ( D ) E14 . 5midgut , ( F ) E14 . 5hind gut , ( H ) postnatal day 14 SI , ( J ) postnatal day 14 colon , ( K ) adult SI . Controls for X ‐ gal staining are shown as inserts ( D ' , F ' , H ' , J ' ) . L , M , N Double immunostaining of small intestinalcrypts for Snai1 / 2 ( red ) and ( M ) Lgr5 ‐ GFP ( green ) ; ( N ) merge shows Snai1 is present in both CBCstem cells and proliferating transit amplifying cells . Scale bars : 10 mm . O , P Immunohistochemistry for Snai1 using a different primary antibody ( Genetex GTX125918 ) shows minimal expression of Snai1 in villi . ( P ) Expression of Snai1 is detected at the base of crypts ( P ) with strong nuclear expression in CBCstem cells indicated by arrows . Scale bars : 20 mm ( O ) , 10 mm ( P ) . Q Schematic diagram showing Snai1 expression ( red ) within different cell types in the crypt . SI = small intestine . See also Supplementary Fig S1 .

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Figure 1A, B Temporal expression of Snail genes in the intestinal tract in (A) embryonic E12.5 to E18.5 and (B) postnatal P0 to P20 measured by qRT-PCR. Shown are means ± SD.C, E, G, I Immunohistochemical staining for Snai1/2 (Abcam ab85931) (C) E14.5midgut, (E) E14.5hind gut, (G) postnatal day 14 SI, (I) postnatal day 14 colon. Snai1immunohistochemistry peptide competition controls are shown as inserts (C', E', G', I'). Scale bars: 20 mm.D, F, H, J, K X‐gal staining for β‐galactosidase activity in tissue from Snai2LacZ mice stained in whole mount, sectioned and counterstained with nuclear fast red (D) E14.5midgut, (F) E14.5hind gut, (H) postnatal day 14 SI, (J) postnatal day 14 colon, (K) adult SI. Controls for X‐gal staining are shown as inserts (D', F', H', J').L, M, N Double immunostaining of small intestinalcrypts for Snai1/2 (red) and (M) Lgr5‐GFP (green); (N) merge shows Snai1 is present in both CBCstem cells and proliferating transit amplifying cells. Scale bars: 10 mm.O, P Immunohistochemistry for Snai1 using a different primary antibody (Genetex GTX125918) shows minimal expression of Snai1 in villi. (P) Expression of Snai1 is detected at the base of crypts (P) with strong nuclear expression in CBCstem cells indicated by arrows. Scale bars: 20 mm (O), 10 mm (P).Q Schematic diagram showing Snai1 expression (red) within different cell types in the crypt. SI = small intestine. See also Supplementary Fig S1.


Figure 2Periodic Acid Schiff ( PAS ) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice . Graph shows number of goblet cells per crypt - villus axes ( n = 3 ) . Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells ( marked by arrows ) . Quantification of enteroendocrine cells per crypt - villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss ( n = 3 , P = 0 . 004 ) . Immunohistochemistry for lysozyme was used to detect Paneth cells . Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 ( n = 4 , P = 0 . 002 ) . Measurement of villus length ( μm ) ( n = 3 , P = 0 . 013 ) and quantification of enterocytes per crypt - villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss ( n = 3 , P = 0 . 036 ) . Measurement of villus length ( μm ) ( n = 3 , P = 0 . 003 ) 8 days after β ‐ NF induction demonstrates a significant shortening of villi . Bars represent mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 20 μm .

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Figure 2Periodic Acid Schiff (PAS) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice. Graph shows number of goblet cells per crypt-villus axes (n = 3).Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells (marked by arrows). Quantification of enteroendocrine cells per crypt-villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss (n = 3, P = 0.004).Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 (n = 4, P = 0.002).Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt-villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.


Figure 3Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells ( arrows depicting proliferating CBC stem cells at the base of crypts ) . Quantification of total PCNA ‐ positive cells per crypt and PCNA ‐ positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 ( n = 4 , P = 0 . 049 , 0 . 026 ) . Scale bars : 20 μm . The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine . Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 ( high magnification inserts depict a representative crypt demarcated by a dotted line ) . Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry . Scale bars : 20 μm . The analysis of sorted and EGFP ‐ positive cells from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1fl / fl and Lgr5 ‐ EGFP ‐ IRES ‐ creERT2AhCreSnai1 + / + mice 5 days after βNF induction show an ˜ tenfold reduction in Lgr5GFP ‐ positive CBCstem cells . Immunohistochemistry for active caspase ‐ 3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt ( n = 5 , P = 0 . 0001 ) and villus ( n = 5 , P = 0 . 005 ) regions in Snai1KO tissue ( arrows mark active caspase ‐ 3 ‐ positive cells ) . Scale bars : 20 μm . A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1 . Tissue from Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1 + / + ( control ) and Lgr5 ‐ EGFP ‐ IRES ‐ creERT2Snai1fl / fl ( Snai1KO ) that also contained the ROSA26LacZCre reporter allele was stained with X ‐ Gal 5 days after tamoxifen injection . Quantification of X ‐ Gal ‐ positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells . Bars represent mean ± SD ( n = 3 , P = 0 . 0001 ) . Scale bars : 0 . 50 mm . Two ‐ tailed Student ' s t ‐ test was used to assess significance . See also Supplementary Figure S2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts). Quantification of total PCNA‐positive cells per crypt and PCNA‐positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 (n = 4, P = 0.049, 0.026). Scale bars: 20 μm.The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine. Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 (high magnification inserts depict a representative crypt demarcated by a dotted line). Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry. Scale bars: 20 μm.The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1+/+mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5GFP‐positive CBCstem cells.Immunohistochemistry for active caspase‐3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt (n = 5, P = 0.0001) and villus (n = 5, P = 0.005) regions in Snai1KO tissue (arrows mark active caspase‐3‐positive cells). Scale bars: 20 μm.A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.


Figure 4VillinCreERT2Snai1fl / florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls . Scale bars : 50 μm . Quantification of viable cell growth in VillinCreERT2Snai1fl / florganoids treated with tamoxifen compared to controls over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 . Assessment of apoptosis in control and VillinCreERT2 Snai1fl / fl organoids after 4 days of growth . Photographs show an overlay of bright ‐ field and fluorescent images with propidium iodide and Hoechst staining . Quantification of apoptotic organoids . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 00001 . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 50 μm . See also Supplementary Figure S3 .

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Figure 4VillinCreERT2Snai1fl/florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cell growth in VillinCreERT2Snai1fl/florganoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.Assessment of apoptosis in control and VillinCreERT2 Snai1fl/fl organoids after 4 days of growth. Photographs show an overlay of bright‐field and fluorescent images with propidium iodide and Hoechst staining. Quantification of apoptotic organoids. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.00001. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 50 μm. See also Supplementary Figure S3.


Figure 5The consequences of Snai1 knockout were analysed over a time course . Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control . The epithelium is re ‐ populated by wild ‐ type cells as shown on day 60 . Scale bars : 20 μm .

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Figure 5The consequences of Snai1 knockout were analysed over a time course. Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control. The epithelium is re‐populated by wild‐type cells as shown on day 60. Scale bars: 20 μm.


Figure 6A PAS staining was used to detect goblet cells in the small intestine , and no differences in cell numbers were observed in test versus control mice ( n = 4 , NS ) . B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells . Quantification of the number of enteroendocrine cells per crypt - villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated ( n = 4 , P = 0 . 003 ) . C Immunohistochemistry for lysozyme was used to detect Paneth cells . Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels ( n = 5 , P = 0 . 027 ) . D , E Quantification of total PCNA ‐ positive cells per crypt revealed a significant increase when Snai1 levels were elevated ( n = 4 , P = 0 . 009 ) . This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of ( E ) PCNA ‐ positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue ( n = 4 , P = 0 . 0005 ) . Bars represent mean ± SD . D , E Quantification of total PCNA ‐ positive cells per crypt revealed a significant increase when Snai1 levels were elevated ( n = 4 , P = 0 . 009 ) . This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of ( E ) PCNA ‐ positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue ( n = 4 , P = 0 . 0005 ) . Bars represent mean ± SD . F PCNAimmunostaining ( green ) and DAPIstaining ( blue ) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates . An expansion of proliferating CBCcells ( indicated by arrows ) is observed at the base of crypts . Scale bars : 10 μm . G qRT - PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates . Bars represent mean ± SD . n = 3 experiments per group , Olfm4 P = 0 . 013 , Cdh1 / E ‐ cadherin P = 0 . 003 , Lgr5 P = 0 . 013 . H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue . A significant increase in CD44v6 ‐ positive cells localised at the base of crypts ( depicted by arrows ) was observed . Bars represent mean ± SD . n = 3 mice per group , * P = 0 . 00023 . Two ‐ tailed Student ' s t ‐ test was used to assess significance . Scale bars : 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A PAS staining was used to detect goblet cells in the small intestine, and no differences in cell numbers were observed in test versus control mice (n = 4, NS).B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells. Quantification of the number of enteroendocrine cells per crypt-villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated (n = 4, P = 0.003).C Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels (n = 5, P = 0.027).D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.F PCNAimmunostaining (green) and DAPIstaining (blue) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates. An expansion of proliferating CBCcells (indicated by arrows) is observed at the base of crypts. Scale bars: 10 μm.G qRT-PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates. Bars represent mean ± SD. n = 3 experiments per group, Olfm4 P = 0.013, Cdh1/E‐cadherin P = 0.003, Lgr5 P = 0.013.H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue. A significant increase in CD44v6‐positive cells localised at the base of crypts (depicted by arrows) was observed. Bars represent mean ± SD. n = 3 mice per group, *P = 0.00023. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 10 μm.


Figure 7VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls . Scale bars : 50 μm . Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days . Bars represent mean ± SD . n = 3 experiments per group , * * P = 0 . 002 . Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth . Photographs show an overlay of bright ‐ field , propidium iodide and Hoechst fluorescence . Scale bars : 50 μm . Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD . n = 3 experiments per group , * * P = 0 . 00003 . Two ‐ tailed Student ' s t ‐ test was used to assess significance .

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Figure 7VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth. Photographs show an overlay of bright‐field, propidium iodide and Hoechst fluorescence. Scale bars: 50 μm.Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD. n = 3 experiments per group, **P = 0.00003. Two‐tailed Student's t‐test was used to assess significance.


Figure 8A - C Droplet digital PCR analysis of Bmf , Bbc3 / Puma and Bcl2l 11 / Bim after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) and VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 h with tamoxifen . No differences in gene expression were observed ( n = 3 ) . D qRT - PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture . VillinCreERT2 Snai1 + / + ( control ) and VillinCreERT2 Snai1fl / fl SI organoids were treated for 24 ( P = 0 . 0001 ) and 72 h ( P = 0 . 006 ) with tamoxifen . Bars represent mean ± SD . n = 3 mice per group . E SW480 cells , which transiently express GFP ‐ SNAI1 , were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1 , IL8 and a novel target SERINC3 revealed by microarray analysis . An unrelated DNA region was used as a negative control . Anti ‐ GFP antibody was used to immunoprecipitate GFP ‐ tagged SNAI1 , while IgG control antibody was used as a negative control . The results are given as relative enrichment compared to input material and are representative of three independent experiments .

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Figure 8A-C Droplet digital PCR analysis of Bmf, Bbc3/Puma and Bcl2l 11/Bim after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 h with tamoxifen. No differences in gene expression were observed (n = 3).D qRT-PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 (P = 0.0001) and 72 h (P = 0.006) with tamoxifen. Bars represent mean ± SD. n = 3 mice per group.E SW480 cells, which transiently express GFP‐SNAI1, were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1, IL8 and a novel target SERINC3 revealed by microarray analysis. An unrelated DNA region was used as a negative control. Anti‐GFP antibody was used to immunoprecipitate GFP‐tagged SNAI1, while IgG control antibody was used as a negative control. The results are given as relative enrichment compared to input material and are representative of three independent experiments.


Figure 1Metaphysis region in the BM is rich in endomucin + capillaries expressing HO - 1 . mp - metaphysis ; gp - growth plate ; scale bar 100 μm . The HO - 1 positive small capillaries in metaphysis express endomucin and CD31 . Shown maximum intensity projection , scale bar 20 μm . HO - 1 is expressed by smaller endomucin + CD31 + capillaries ( # ) as well as in bigger endomucin - / lowCD31 + arteries ( * ) . CD31 - pericytes wrapping the artery also express HO - 1 ( * ) ; scale bar 20 μm . HO - 1 positive capillaries in the metaphysis expressed CD31 and Sca - 1 . The capillaries are enveloped by HO - 1 expressing pericytes . Part of the HO - 1 + pericytes express Sca - 1 ( # ) while others show no or low Sca - 1 signal ( * ) ; scale bar 20 μm . Flow cytometry analysis revealed the highest expression of HO - 1 in CD31 + Sca - 1 + ECs . CAR and PαS populations also express HO - 1 , while most of non - hematopoietic CD45 - Ter119 - are HO - 1 - negative in steady - state conditions . BM macrophages ( MQ ) express HO - 1 . The MHCIIhigh MQ express higher levels of HO - 1 than MHCIIlow MQ . Cells within whole HSPC compartment ( LKS ) express no or low levels of HO - 1 in comparison to MQ . HO - 1 expression on mRNA level quantified by qPCR qPCR analysis based on two independent experiments n = 10 - 11 / groupHO - 1 expression on mRNA level quantified by RNA - seq . RNAseq analysis has n = 3 - 4 / group , two - tailed t - test for two groups comparison , and one way ANOVA with Bonferroni post - test for multiple group comparison . * p < 0 . 05 , * * * * p < 0 . 0001 . Data are shown as mean ± SEM . ECs and CARs from middle - aged animals ( 11 - 12 months ) express lower levels of HO - 1 protein . Two independent experiments , n = 5 - 10 / group . Data are shown as mean ± SEM . * p < 0 . 05 , * * * p < 0 . 001 , two - tailed unpaired t - test . Middle - aged animals have lower frequency of ECs . Two independent experiments , n = 10 - 11 / group . Data are shown as mean ± SEM . * * p < 0 . 01 , two - tailed unpaired t - test . The control staining of HO - 1 on HO - 1 - / - bone marrow section is provided in Appendix Fig S1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Metaphysis region in the BM is rich in endomucin+ capillaries expressing HO-1. mp - metaphysis; gp - growth plate; scale bar 100 μm. The HO-1 positive small capillaries in metaphysis express endomucin and CD31. Shown maximum intensity projection, scale bar 20 μm. HO-1 is expressed by smaller endomucin+CD31+ capillaries (#) as well as in bigger endomucin-/lowCD31+ arteries (*). CD31- pericytes wrapping the artery also express HO-1 (*); scale bar 20 μm. HO-1 positive capillaries in the metaphysis expressed CD31 and Sca-1. The capillaries are enveloped by HO-1 expressing pericytes. Part of the HO-1+ pericytes express Sca-1 (#) while others show no or low Sca-1 signal (*); scale bar 20 μm.Flow cytometry analysis revealed the highest expression of HO-1 in CD31+Sca-1+ ECs. CAR and PαS populations also express HO-1, while most of non-hematopoietic CD45-Ter119- are HO-1-negative in steady-state conditions.BM macrophages (MQ) express HO-1. The MHCIIhigh MQ express higher levels of HO-1 than MHCIIlow MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO-1 in comparison to MQ.HO-1 expression on mRNA level quantified by qPCR qPCR analysis based on two independent experiments n = 10-11/groupHO-1 expression on mRNA level quantified by RNA-seq. RNAseq analysis has n = 3-4/group, two-tailed t-test for two groups comparison, and one way ANOVA with Bonferroni post-test for multiple group comparison. *p< 0.05, ****p< 0.0001. Data are shown as mean ± SEM.ECs and CARs from middle-aged animals (11-12 months) express lower levels of HO-1 protein. Two independent experiments, n = 5-10/group. Data are shown as mean ± SEM. *p< 0.05, ***p<0.001, two-tailed unpaired t-test.Middle-aged animals have lower frequency of ECs. Two independent experiments, n = 10-11/group. Data are shown as mean ± SEM. **p<0.01, two-tailed unpaired t-test. The control staining of HO-1 on HO-1-/- bone marrow section is provided in Appendix Fig S1.


Figure 2RNA - seq revealed 111 DEGs in HO - 1 - / - ECs . The identified DEGs separated HO - 1 - / - and HO - 1 + / + ECs by ( B ) hierarchical clustering and ( C ) PCA . Heatmap of selected ( E ) hematopoietic factors and ( F ) adhesion molecules expression in HO - 1 - / - and HO - 1 + / + ECs . RNA - seq revealed 100 DEGs in HO - 1 - / - CARs . The identified DEGs separated HO - 1 - / - and HO - 1 + / + CARs by ( H ) hierarchical clustering and ( I ) PCA . Heatmap of selected ( K ) skeletal biology and ( L ) hematopoietic factors expression in HO - 1 - / - and HO - 1 + / + CARs . Correlation similarity metric and average linkage clustering were used in the presented heatmaps .

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Figure 2RNA-seq revealed 111 DEGs in HO-1-/- ECs.The identified DEGs separated HO-1-/- and HO-1+/+ ECs by (B) hierarchical clustering and (C) PCA.Heatmap of selected (E) hematopoietic factors and (F) adhesion molecules expression in HO-1-/- and HO-1+/+ ECs.RNA-seq revealed 100 DEGs in HO-1-/- CARs.The identified DEGs separated HO-1-/- and HO-1+/+ CARs by (H) hierarchical clustering and (I) PCA.Heatmap of selected (K) skeletal biology and (L) hematopoietic factors expression in HO-1-/- and HO-1+/+ CARs. Correlation similarity metric and average linkage clustering were used in the presented heatmaps.


Figure 3HO - 1 - / - mice have more ECs and less CARs ( p = 0 . 053 ) in BM , while frequency of PaS does not differ . Young HO - 1 - / - mice have lower frequency of Sca - 1high fraction among BM ECs , what resembles phenotype of BM ECs in old mice . Intracellular flow cytometry reveals ( C ) lower levels of SDF - 1α in HO - 1 - / - CARs and ( D ) lower levels of LAP protein in HO - 1 - / - ECs . Analysis of cell surface SCF revealed lower frequency of SCF + ECs in HO - 1 - / - while no differences among CARs . The frequency of ( F ) CD163 + MQs and ( G ) CD163 - MQs in BM of HO - 1 - / - mice is altered .

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Figure 3HO-1-/- mice have more ECs and less CARs (p = 0.053) in BM, while frequency of PaS does not differ.Young HO-1-/- mice have lower frequency of Sca-1high fraction among BM ECs, what resembles phenotype of BM ECs in old mice.Intracellular flow cytometry reveals (C) lower levels of SDF-1α in HO-1-/- CARs and (D) lower levels of LAP protein in HO-1-/- ECs.Analysis of cell surface SCF revealed lower frequency of SCF+ ECs in HO-1-/- while no differences among CARs.The frequency of (F) CD163+ MQs and (G) CD163- MQs in BM of HO-1-/- mice is altered.


Figure 4Gating strategy of hematopoietic stem and progenitor cells . Young HO - 1 - / - mice possess higher number of ( B ) LT - HSCs , ( C ) ST - HSCs and ( D ) MPPs . Exemplary analysis of cell cycle with Ki67 and nuclear dye . More young HO - 1 - / - LT - HSCs are in G1 and S / G2 / M cell cycle phases . The presented cell cycle analysis is from two independent experiments . Young HO - 1 - / - MPPs do not differ in cell cycling from young HO - 1 + / + MPPs . The presented cell cycle analysis is from two independent experiments . More old HO - 1 - / - LT - HSCs are in G1 phase in comparison to old HO - 1 + / + LT - HSCs , but not in S / G2 / M phase . The presented cell cycle analysis is from two independent experiments . Old MPPs do not differ in cell cycling between genotypes . The presented cell cycle analysis is from two independent experiments . Young HO - 1 - / - LT - HSCs contain more γH2aXhigh cells than young HO - 1 + / + , 378 - 991 cells analyzed from 7 mice / group . Young HO - 1 - / - MPPs contain more γH2aXhigh cells than young HO - 1 + / + , 1693 - 2790 cells analyzed from 7 mice / group . Alkaline comet assay revealed that HO - 1 - / - LT - HSCs possess ( L ) higher oil tail moment and ( M ) more DNA in the comet tail . 1068 - 1189 cells analyzed from 6 mice / group . HSCs from young HO - 1 - / - mice provide worse hematopoietic reconstitution after transplantation than HO - 1 + / + HSCs . Data are shown as mean ± SEM , n = 8 - 9 mice / group .

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Figure 4Gating strategy of hematopoietic stem and progenitor cells.Young HO-1-/- mice possess higher number of (B) LT-HSCs, (C) ST-HSCs and (D) MPPs.Exemplary analysis of cell cycle with Ki67 and nuclear dye.More young HO-1-/- LT-HSCs are in G1 and S/G2/M cell cycle phases. The presented cell cycle analysis is from two independent experiments. Young HO-1-/- MPPs do not differ in cell cycling from young HO-1+/+ MPPs. The presented cell cycle analysis is from two independent experiments. More old HO-1-/- LT-HSCs are in G1 phase in comparison to old HO-1+/+ LT-HSCs, but not in S/G2/M phase. The presented cell cycle analysis is from two independent experiments. Old MPPs do not differ in cell cycling between genotypes. The presented cell cycle analysis is from two independent experiments.Young HO-1-/- LT-HSCs contain more γH2aXhigh cells than young HO-1+/+, 378-991 cells analyzed from 7 mice/group. Young HO-1-/- MPPs contain more γH2aXhigh cells than young HO-1+/+, 1693-2790 cells analyzed from 7 mice/group.Alkaline comet assay revealed that HO-1-/- LT-HSCs possess (L) higher oil tail moment and (M) more DNA in the comet tail. 1068-1189 cells analyzed from 6 mice/group.HSCs from young HO-1-/- mice provide worse hematopoietic reconstitution after transplantation than HO-1+/+ HSCs. Data are shown as mean ± SEM, n = 8-9 mice/group.


Figure 5RNA - seq revealed 1067 DEGs between young HO - 1 - / - and HO - 1 + / + LT - HSCs . RNA - seq revealed 595 DEGs between old HO - 1 - / - and HO - 1 + / + LT - HSCs . PC1 in PCA analysis on selected 1148 genes separated young HO - 1 + / + LT - HSCs from other groups , clustering the young HO - 1 - / - LT - HSCs together with old LT - HSCs . Hierarchical clustering on the 20 % of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO - 1 + / + LT - HSCs in one and old LT - HSCs with young HO - 1 - / - LT - HSCs in second .

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Figure 5RNA-seq revealed 1067 DEGs between young HO-1-/- and HO-1+/+ LT-HSCs. RNA-seq revealed 595 DEGs between old HO-1-/- and HO-1+/+ LT-HSCs.PC1 in PCA analysis on selected 1148 genes separated young HO-1+/+ LT-HSCs from other groups, clustering the young HO-1-/- LT-HSCs together with old LT-HSCs.Hierarchical clustering on the 20% of most correlated 297 genes with PC1 showed 2 major clusters that includes young HO-1+/+ LT-HSCs in one and old LT-HSCs with young HO-1-/- LT-HSCs in second.


Figure 6Deletion of HO - 1 in myeloid lineage did not cause expansion of LT - HSCs and did not alter their cell cycle status . Long - term PB chimerism derived from HSCs transplanted to HO - 1 - / - recipients is lower than from HSCs transplanted to HO - 1 + / + recipients . Chimerism among BM HSPCs fractions transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± SEM , n = 5 - 12 / grouptotal number of cells derived from HSCs transplanted to HO - 1 - / - recipients are lower than from HSCs transplanted to HO - 1 + / + recipients . Data are shown as mean ± SEM , n = 5 - 12 / groupShort term homing of HSPC to HO - 1 + / + or HO - 1 - / - BM did not differ . Data are shown as mean ± SEM , n = 5 / group , unpaired t - test .

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Figure 6Deletion of HO-1 in myeloid lineage did not cause expansion of LT-HSCs and did not alter their cell cycle status.Long-term PB chimerism derived from HSCs transplanted to HO-1-/- recipients is lower than from HSCs transplanted to HO-1+/+ recipients.Chimerism among BM HSPCs fractions transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/grouptotal number of cells derived from HSCs transplanted to HO-1-/- recipients are lower than from HSCs transplanted to HO-1+/+ recipients. Data are shown as mean ± SEM, n = 5-12/groupShort term homing of HSPC to HO-1+/+ or HO-1-/- BM did not differ. Data are shown as mean ± SEM, n = 5/group, unpaired t-test.


Figure 7Transplantation of HO - 1 - / - HSCs provides lower chimerism among HSPC fractions in primary recipients . Data are shown as mean ± SEM , n = 7 - 8 mice / group . Transplantation of the same number of donor - derived BM cells from primary recipients provides the same ( C ) PB chimerismTransplantation of the same number of donor - derived BM cells from primary recipients provides the same BM LT - HSC chimerism . Comparison of gene log fold changes in non - transplanted HO - 1 - / - LT - HSCs and HO - 1 - / - LT - HSCs transplanted twice to the wild - type HO - 1 + / + showed that transplantation of HO - 1 - / - LT - HSCs twice to the wild type niche reverses their transcriptional alterations .

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Figure 7Transplantation of HO-1-/- HSCs provides lower chimerism among HSPC fractions in primary recipients. Data are shown as mean ± SEM, n = 7-8 mice/group.Transplantation of the same number of donor-derived BM cells from primary recipients provides the same (C) PB chimerismTransplantation of the same number of donor-derived BM cells from primary recipients provides the same BM LT-HSC chimerism.Comparison of gene log fold changes in non-transplanted HO-1-/- LT-HSCs and HO-1-/- LT-HSCs transplanted twice to the wild-type HO-1+/+ showed that transplantation of HO-1-/- LT-HSCs twice to the wild type niche reverses their transcriptional alterations.


Figure 8Frequency of colonies appearing at day 8 and day 12 from HSCs co - cultured with HO - 1 + / + or HO - 1 - / - MSCs . * p < 0 . 05 , * * * p < 0 . 001 , Fisher exact test . PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed ( C ) 4 distinct clusters of colonies , that differ by frequency of ( D ) CD11b + Gr1 - , ( E ) CD11b + Gr1 + 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 . 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 / groupLT - 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 / groupcell cycling status of LT - HSCs after administration of Noggin to HO - 1 + / + or HO - 1 - / - mice .

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Figure 8Frequency of colonies appearing at day 8 and day 12 from HSCs co-cultured with HO-1+/+ or HO-1-/- MSCs. *p< 0.05, ***p<0.001, Fisher exact test.PCA and cluster analysis of the colonies based on initial flow cytometry gating revealed (C) 4 distinct clusters of colonies, that differ by frequency of (D) CD11b+Gr1-, (E) CD11b+Gr1+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.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/groupLT-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/groupcell cycling status of LT-HSCs after administration of Noggin to HO-1+/+ or HO-1-/- mice.


Figure 1Media 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 timesMedia 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 timesMedia 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 6E10Media 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 -

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Figure 1Media 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 timesMedia 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 timesMedia 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 6E10Media 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


Figure 2Schematic 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 =

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Figure 2Schematic 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


Figure 3 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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Figure 3 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


Figure 4 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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Figure 4 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)


Figure 52B4 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 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 timesHEK293T 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 52B4 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 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 timesHEK293T 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


figf1 ( 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 . ( 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 . ( 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 ) . ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(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.(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.(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).(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.


figf2 ( 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 . ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(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.(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.


figf3 ( 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 . ( 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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figf3(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.(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).