Figure 4Immunoblo of mGPD in GA muscles from obese patientIH of mGPD in GA muscles from obese patientImmunoblo of mGPDH and myogenin in GA muscles fro indicated micqRT - PCR of mGPDH in GA muscle of indicated mice at days 0 and 3 post CTX intramuscular injectionExperimental setup ( upper of E ) , qRT - PCR of mGPDH , myogenin and myh3 ( bottom of E in GA muscle from AAV - mGPDH - treated HFD - fed mice at days 7 post CTX intramuscular injection ) , H & E staining ( arrowhead , necrotic myofibers ; asterisks , regenerating fibers ) ( F ) , distribution of the fiber CSA ( G ) in GA muscle from AAV - mGPDH - treated HFD - fed mice at days 7 post CTX intramuscular injectionpercentage of myofibers with central nuclei ( H ) in GA muscle from AAV - mGPDH - treated HFD - fed mice at days 7 post CTX intramuscular injectionExperimental setup ( upper of I qRT - PCR of mGPDH , myogenin and myh3 ( bottom of I ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post CTX intramuscular injectionH & E staining ( arrowhead , necrotic myofibers ; asterisks , regenerating fibers ) ( J ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post CTX intramuscular injectiondistribution of the fibers CSA ( K ) , percentage of myofibers with central nuclei ( L in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post CTX intramuscular injectionExperimental setup ( uppe and muscle weight ( bottom of M ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at da 14 ( M ) post CTX intramuscular injectionImmunoblots of mGPDH , p - AMPK , p - ACC , PGC1α and myogenin for the experiment described in E .

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

Figure 4Immunoblo of mGPD in GA muscles from obese patientIH of mGPD in GA muscles from obese patientImmunoblo of mGPDH and myogenin in GA muscles fro indicated micqRT-PCR of mGPDH in GA muscle of indicated mice at days 0 and 3 post CTX intramuscular injectionExperimental setup (upper of E), qRT-PCR of mGPDH, myogenin and myh3 (bottom of E in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection), H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (F), distribution of the fiber CSA (G) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injectionpercentage of myofibers with central nuclei (H) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injectionExperimental setup (upper of I qRT-PCR of mGPDH, myogenin and myh3 (bottom of I) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injectionH&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (J) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injectiondistribution of the fibers CSA (K), percentage of myofibers with central nuclei (L in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injectionExperimental setup (uppe and muscle weight (bottom of M) in GA muscle from AAV-mGPDH-treated STZ-treated mice at da 14 (M) post CTX intramuscular injectionImmunoblots of mGPDH, p-AMPK, p-ACC, PGC1α and myogenin for the experiment described in E.


Figure 1A ) Left : Representative current traces from wildtype NavMs channels showing the first 0 . 1 s of 0 . 5 s activations from - 180 mV holding potential recorded with the indicated extracellular cations . Right : Resulting current - voltage profiles profiles , where current magnitudes were normalized to the maximum inward current measured in the sodium condition . B ) Left : Representative current traces from mutant channel E178D ( as in A ) . Right : Resulting current - voltage profiles ( error ± SEM ; n = 6 - 9 for NavMs ; n = 4 - 8 for E178D ) .

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

Figure 1A) Left: Representative current traces from wildtype NavMs channels showing the first 0.1 s of 0.5 s activations from -180 mV holding potential recorded with the indicated extracellular cations. Right: Resulting current-voltage profiles profiles, where current magnitudes were normalized to the maximum inward current measured in the sodium condition.B) Left: Representative current traces from mutant channel E178D (as in A). Right: Resulting current-voltage profiles (error ± SEM; n = 6-9 for NavMs; n = 4-8 for E178D).


Figure 2A ) Upper : Overview of the AB tetramer , with each of the monomers depicted in a different colour . The front monomer has been removed for clarity . The black box indicates the region expanded in the lower panel . Lower : An expanded view of the selectivity filter , showing only two monomers as pink and white ribbon cartoons with the selectivity filter residues drawn as sticks in the corresponding colours . Sodium ions are shown as cyan spheres and well - ordered waters as red spheres ( at 0 . 4x their actual radii , for clarity ) . The final refined 2Fo - Fc electron density maps for the protein , ions and water are shown at 2 . 0 σ in dark grey mesh and 0 . 8 σ in light grey , and the initial difference map ( without sodiums or waters included in the phase calculation ) at 3 . 0 σ in green mesh . B ) Superposition of the sodium ion binding sites found in the crystallographically - distinct AB and CD tetramers and the proposed binding sites from other studies . The tetramers were superposed with the LSQKAB program ( Kabsch , 2010 ) from CCP4 ( Winn et al . , 2011 ) using residues 131 - 220 from all four monomers in each tetramer . Only two monomers from the AB tetramer are shown for clarity , and coloured as in panel A . Sodium ions from the AB tetramer are depicted as cyan spheres , those from the CD tetramer as teal spheres . Predicted sodium ion binding sites from Ulmschneider et al . ( 2013 ) are indicated as small black spheres and labelled on the right of the spheres according to the nomenclature ( S0 to S4 ) in that study . Predicted sodium ion binding sites from Payendeh et al . ( 2011 ) , based on a closed channel structure of the close orthologue NavAb , are indicated on the far right and labelled as per the nomenclature of that study ( SiteHFS , SiteCEN and SiteIN ) . This and subsequent molecular figures were drawn with PyMol ( The PyMOL Molecular Graphics System , Version 1 . 6 , Schrödinger , LLC ) unless otherwise stated .

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

Figure 2A) Upper: Overview of the AB tetramer, with each of the monomers depicted in a different colour. The front monomer has been removed for clarity. The black box indicates the region expanded in the lower panel. Lower: An expanded view of the selectivity filter, showing only two monomers as pink and white ribbon cartoons with the selectivity filter residues drawn as sticks in the corresponding colours. Sodium ions are shown as cyan spheres and well-ordered waters as red spheres (at 0.4x their actual radii, for clarity). The final refined 2Fo-Fc electron density maps for the protein, ions and water are shown at 2.0 σ in dark grey mesh and 0.8 σ in light grey , and the initial difference map (without sodiums or waters included in the phase calculation) at 3.0 σ in green mesh.B) Superposition of the sodium ion binding sites found in the crystallographically-distinct AB and CD tetramers and the proposed binding sites from other studies. The tetramers were superposed with the LSQKAB program (Kabsch, 2010) from CCP4 (Winn et al., 2011) using residues 131-220 from all four monomers in each tetramer. Only two monomers from the AB tetramer are shown for clarity, and coloured as in panel A. Sodium ions from the AB tetramer are depicted as cyan spheres, those from the CD tetramer as teal spheres. Predicted sodium ion binding sites from Ulmschneider et al. (2013) are indicated as small black spheres and labelled on the right of the spheres according to the nomenclature (S0 to S4) in that study. Predicted sodium ion binding sites from Payendeh et al. (2011), based on a closed channel structure of the close orthologue NavAb, are indicated on the far right and labelled as per the nomenclature of that study (SiteHFS, SiteCEN and SiteIN). This and subsequent molecular figures were drawn with PyMol (The PyMOL Molecular Graphics System, Version 1.6, Schrödinger, LLC) unless otherwise stated.


Figure 3A ) Top : Crystal structure overview of the wildtype AB tetramer ( PDBID 5ZBZ ) , view as in Figure 2A . Left : Difference ( Fo - Fc ) electron density map ( mutant minus wildtype ) contoured at - 2 . 5σ and drawn as red mesh , plotted on the structure of the wildtype ( E178 ) protein , highlighting the consequences of the loss of the δ - carbon atom from residue 178 in the E178D mutant . For reference , the locations of the sodium ion sites present in the wildtype ( E178 ) structure are depicted as cyan spheres . Both the structure and the ions are displayed as faded backgrounds so that the difference map can be seen more clearly . Right : Final refined 2Fo - Fc electron density map for the E178D mutant ( PDBID 4X88 ) , contoured at 1 . 5σ and shown as grey mesh , with the sodium ions depicted as cyan spheres . B ) Surface representations of ( left ) wildtype NavMs pore and ( right ) the E178D mutant coloured by electrostatic charge calculated by APBS ( Callenberg et al , 2010 ) and drawn with VMD ( Humphrey et al . , 1996 ) , showing a slice through the middle of the tetramer . Sodium ions are shown as cyan spheres ; only 2 are present in the mutant , instead of the 3 seen in the wildtype , with the top sodium missing in the mutant . The lower panels are expanded views of the selectivity filter regions , with the E178 side chains depicted in yellow sticks and the location of the main chain nitrogen of residue 179 indicated by the black circle . The alteration in the protein structure caused by removal of the methylene group of E178 exposes a positive region of the polypeptide backbone adjacent to the site where the top sodium ion is located in the wildtype protein ; this likely contributes to why an equivalent sodium ion is not seen adjacent to this position in the mutant .

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

Figure 3A) Top: Crystal structure overview of the wildtype AB tetramer (PDBID 5ZBZ), view as in Figure 2A.Left: Difference (Fo-Fc) electron density map (mutant minus wildtype) contoured at -2.5σ and drawn as red mesh, plotted on the structure of the wildtype (E178) protein, highlighting the consequences of the loss of the δ-carbon atom from residue 178 in the E178D mutant. For reference, the locations of the sodium ion sites present in the wildtype (E178) structure are depicted as cyan spheres. Both the structure and the ions are displayed as faded backgrounds so that the difference map can be seen more clearly.Right: Final refined 2Fo-Fc electron density map for the E178D mutant (PDBID 4X88), contoured at 1.5σ and shown as grey mesh, with the sodium ions depicted as cyan spheres.B) Surface representations of (left) wildtype NavMs pore and (right) the E178D mutant coloured by electrostatic charge calculated by APBS (Callenberg et al, 2010) and drawn with VMD (Humphrey et al., 1996), showing a slice through the middle of the tetramer. Sodium ions are shown as cyan spheres; only 2 are present in the mutant, instead of the 3 seen in the wildtype, with the top sodium missing in the mutant. The lower panels are expanded views of the selectivity filter regions, with the E178 side chains depicted in yellow sticks and the location of the main chain nitrogen of residue 179 indicated by the black circle. The alteration in the protein structure caused by removal of the methylene group of E178 exposes a positive region of the polypeptide backbone adjacent to the site where the top sodium ion is located in the wildtype protein; this likely contributes to why an equivalent sodium ion is not seen adjacent to this position in the mutant.


Figure 4Left : Sodium ions ( cyan ) in the selectivity filter of the NavMs sodium channel . Only two monomers ( in white and pink ) are shown for clarity . Coordinates are from the AB tetramer of PDBID 5BZB . Right : Potassium ions ( blue ) in the selectivity filter of the KcsA potassium channel . As for the NavMs structure , only two monomers ( in grey and maroon ) are shown for clarity . Coordinates are from PDBID 1K4C ( Zhou et al . , 2004 ) . The two crystal structures were first superimposed using SSM ( Krissinel and Henrick , 2004 ) , and are drawn using the same scale , and with the correct ratio between the diameters of the ions used , so that the differences in the internal dimensions of their selectivity filters can be seen , thus making it clear how much more space there is available in the NavMs SF for intervening water molecules . In the KcsA channel , the ions are directly coordinated to the polypeptide backbone atoms , whereas in the NavMs SF , the closest atoms ( side chains ) are obviously at longer distances than direct bonding to the ions , enabling interactions via water molecules .

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

Figure 4Left: Sodium ions (cyan) in the selectivity filter of the NavMs sodium channel. Only two monomers (in white and pink) are shown for clarity. Coordinates are from the AB tetramer of PDBID 5BZB.Right: Potassium ions (blue) in the selectivity filter of the KcsA potassium channel. As for the NavMs structure, only two monomers (in grey and maroon) are shown for clarity. Coordinates are from PDBID 1K4C (Zhou et al., 2004).The two crystal structures were first superimposed using SSM (Krissinel and Henrick, 2004), and are drawn using the same scale, and with the correct ratio between the diameters of the ions used, so that the differences in the internal dimensions of their selectivity filters can be seen, thus making it clear how much more space there is available in the NavMs SF for intervening water molecules. In the KcsA channel, the ions are directly coordinated to the polypeptide backbone atoms, whereas in the NavMs SF, the closest atoms (side chains) are obviously at longer distances than direct bonding to the ions, enabling interactions via water molecules.


Figure 1A . Representative FACS plots of freshly isolated epidermal cells ( Epi d0 ) and day 14 ( d14 ) cultures of epidermal cells in different growth conditions and media . KGM 2D : 2D in basal KGM ; 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . B . CD34 + α6 + cells were quantified by flow cytometry from d14 as in ( A ) ( mean ± SD ; n = 3 - 5 ; * p ≤ 0 . 05 ; * * * p ≤ 0 . 001 , Kruskal - Wallis / Dunn ' s post test ) . 3C 2D : 2D in KGM + Y27632 + VEGF - A + FGF - 2 ; Y : 3D - Matrigel in KGM + Y27632 ; YV : Y + VEGF - A ; YF : Y + FGF - 2 ; 3C : Y + VEGF - A + FGF - 2 . C . Absolute numbers of CD34 + α6 + cells from Epi d0 and d14 cultures as in ( A - B ) . Fold enrichment over Epi d0 is shown ( mean ± SEM , n = 3 - 5 ; * p ≤ 0 . 05 , Mann - Whitney U test ) . D . Epidermal cells grown for 14 days in the indicated conditions . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . 3C 2D : 2D in 3C medium . Scale bars 30µm . E . Immunofluorescence analysis of 3C cultures showing the expression of the HFSC markers CD34 and SOX9 in a subset of cells . See also Appendix Appendix figure 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1A. Representative FACS plots of freshly isolated epidermal cells (Epi d0) and day 14 (d14) cultures of epidermal cells in different growth conditions and media. KGM 2D: 2D in basal KGM; 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. CD34+α6+ cells were quantified by flow cytometry from d14 as in (A) (mean ±SD; n=3-5; *p ≤ 0.05; ***p ≤ 0.001, Kruskal-Wallis/Dunn's post test). 3C 2D: 2D in KGM + Y27632 + VEGF-A + FGF-2; Y: 3D-Matrigel in KGM + Y27632; YV: Y + VEGF-A; YF: Y + FGF-2; 3C: Y + VEGF-A + FGF-2. C. Absolute numbers of CD34+α6+ cells from Epi d0 and d14 cultures as in (A-B). Fold enrichment over Epi d0 is shown (mean ±SEM, n=3-5; *p ≤ 0.05, Mann-Whitney U test).D. Epidermal cells grown for 14 days in the indicated conditions. 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. 3C 2D: 2D in 3C medium. Scale bars 30µm.E. Immunofluorescence analysis of 3C cultures showing the expression of the HFSC markers CD34 and SOX9 in a subset of cells. See also Appendix Appendix figure S1.


Figure 2A . Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells ( Epi d0 ; control ) ( mean ± SEM ; n = 5 ; * p ≤ 0 . 05 , Mann - Whitney U - test ) . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . B . Full thickness skin reconstitution assay with freshly isolated epidermal cells ( Epi d0 ) or 3C cultures shows that cells cultured in 3C retain their self - renewal capacity and multipotency . A representative recipient of 4 mice / condition is shown . Right panel shows quantification of hair follicle density . Note that 3C cultures give rise to more hair compared to controls ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U test ) . C . Long - term culture of cells in 3C was performed as shown in the left panel . 3C cultures maintain a stable population of CD34 + α6 + long term ( data are shown using a Box - and - Whisker plot : box indicates 25th and 75th percentiles ; error bars represent 10th and 90th percentiles ; middle line is the median ; n = 3 - 5 independent experiments ) . p : passage . D . Full thickness skin reconstitution assay using 3C cells from passage 1 ( p1 ) and passage 15 ( p15 ) show that cells cultured in 3C maintain their multipotency and self - renewal capacity in long - term cultures . A representative recipient of 4 mice / condition is shown . See also Appendix 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 2A. Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells (Epi d0; control) (mean ±SEM; n=5; *p ≤ 0.05, Mann-Whitney U-test). 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2.B. Full thickness skin reconstitution assay with freshly isolated epidermal cells (Epi d0) or 3C cultures shows that cells cultured in 3C retain their self-renewal capacity and multipotency. A representative recipient of 4 mice/condition is shown. Right panel shows quantification of hair follicle density. Note that 3C cultures give rise to more hair compared to controls (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).C. Long-term culture of cells in 3C was performed as shown in the left panel. 3C cultures maintain a stable population of CD34+α6+ long term (data are shown using a Box-and-Whisker plot: box indicates 25th and 75th percentiles; error bars represent 10th and 90th percentiles; middle line is the median; n=3-5 independent experiments). p: passage.D. Full thickness skin reconstitution assay using 3C cells from passage 1 (p1) and passage 15 (p15) show that cells cultured in 3C maintain their multipotency and self-renewal capacity in long-term cultures. A representative recipient of 4 mice/condition is shown. See also Appendix figure S2.


Figure 3B . Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A . 3C cultures cluster with purified CD34 + α6 + HFSCs ( n = 3 biological replicates ) . E . Heatmap of selected genes differentially expressed in 3C - CD34 + α6 + and 3C - CD34 ¯ α6 + cells . Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers ( triangles ) in 3C - CD34 + α6 + cells , and upregulation of genes implicated in lineage progression in 3C - CD34 ¯ α6 + cells ( circles ) . G . RT - qPCR analyses of FACS - purified CD34 + α6 + and CD34 - α6 + cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C - CD34 + α6 + cells compared to freshly isolated CD34 - α6 + progenitors . 3C - CD34 - α6 + cells show intermediated expression ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . H . Both 3C - CD34 + α6 + and 3C - CD34 - α6 + cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S3 and Tables S1 - S3 .

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

Figure 3B. Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A. 3C cultures cluster with purified CD34+α6+ HFSCs (n=3 biological replicates).E. Heatmap of selected genes differentially expressed in 3C-CD34+α6+ and 3C-CD34¯α6+ cells. Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers (triangles) in 3C-CD34+α6+ cells, and upregulation of genes implicated in lineage progression in 3C-CD34¯α6+ cells (circles).G. RT-qPCR analyses of FACS-purified CD34+α6+ and CD34-α6+ cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C-CD34+α6+ cells compared to freshly isolated CD34-α6+ progenitors. 3C- CD34-α6+ cells show intermediated expression (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).H. Both 3C-CD34+α6+ and 3C-CD34-α6+ cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S3 and Tables S1-S3.


Figure 4A . FACS analysis of CD34 + α6 + cells over time shows that 3C cultures reach a ~ 50 : 50 population equilibrium after 10 days of culture ( mean ± SEM ; n = 3 ) . B . Cells were EdU - labeled for 24 h ( day 10 - 11 ) prior to analysis by flow cytometry to quantify proliferating cells . Percentage of EdU + cells is indicated ( n = 4 ; mean ± SD ; * p ≤ 0 . 05 , Mann - Whitney U - test ) . D . Simulated population proportions of CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs derived from the mathematical model describing steady state cultures . Experimental data from ( A ) are shown as reference ( in grey ) . See also Appendix figure S4 and Dataset EV4 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A. FACS analysis of CD34+α6+ cells over time shows that 3C cultures reach a ~50:50 population equilibrium after 10 days of culture (mean ±SEM; n=3).B. Cells were EdU-labeled for 24 h (day 10-11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test).D. Simulated population proportions of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs derived from the mathematical model describing steady state cultures. Experimental data from (A) are shown as reference (in grey). See also Appendix figure S4 and Dataset EV4.


Figure 5A . CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs were FACS - purified from total epidermis ( d0 ) to ≥ 98 % purity post sorting ( d0 post - sort ) , and grown 14 days in 3C cultures ( d14 3C ) . A significant population of CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs is generated from both purified populations after 14 days . A representative of 3 independent experiments is shown ( mean ± SD ) . B - C . Time course analyses of 3C cultures established from FACS - purified populations of CD34 + α6 + HFSCs ( B ) or CD34 ¯ α6 + non - HFSCs ( C ) from 3C cultures . Both cultures reach population equilibrium between day 6 and 10 ( n = 4 ; mean ± SD ) . D - E . Simulated population proportions of CD34 + α6 + HFSCs ( D ) and CD34 ¯ α6 + non - HFSCs ( E ) from the mathematical model describing behavior of pure cell populations . The model parameters were estimated from the experimental data in B - C ( shown in grey as reference ) . G . Lineage tracing of FACS - purified CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs from EGFP ¯ and EGFP + 3C cultures , respectively . CD34 + α6 + and CD34 ¯ α6 + were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry . Presence of CD34 + α6 + EGFP + cells in cultures at d10 indicates non - HFSC to HFSC interconversion . H . Lineage tracing as in ( F ) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non - HFSCs . I . Quantification of the proportion of CD34 + α6 + cells that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled cells are indicated ) . J . Quantification of the proportion of CD34 ¯ α6 + cells that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled cells are indicated ) . Data in panels G - J show n = 4 ; mean ± SD . See also Appendix figure S5 .

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Figure 5A. CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs were FACS-purified from total epidermis (d0) to ≥98% purity post sorting (d0 post-sort), and grown 14 days in 3C cultures (d14 3C). A significant population of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs is generated from both purified populations after 14 days. A representative of 3 independent experiments is shown (mean ±SD).B-C. Time course analyses of 3C cultures established from FACS-purified populations of CD34+α6+ HFSCs (B) or CD34¯α6+ non-HFSCs (C) from 3C cultures. Both cultures reach population equilibrium between day 6 and 10 (n=4; mean ±SD).D-E. Simulated population proportions of CD34+α6+ HFSCs (D) and CD34¯α6+ non-HFSCs (E) from the mathematical model describing behavior of pure cell populations. The model parameters were estimated from the experimental data in B-C (shown in grey as reference).G. Lineage tracing of FACS-purified CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs from EGFP¯ and EGFP+ 3C cultures, respectively. CD34+α6+ and CD34¯α6+ were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry. Presence of CD34+α6+ EGFP+ cells in cultures at d10 indicates non-HFSC to HFSC interconversion.H. Lineage tracing as in (F) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non-HFSCs.I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated).J. Quantification of the proportion of CD34¯α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). Data in panels G-J show n=4; mean ±SD. See also Appendix figure S5.


Figure 6A - B . Inhibitor treatment scheme ( A ) and FACS plots ( B ) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin ( mean ± SD ; n = 3 ) . C . Quantification of experiments as in ( B ) shows enrichment of HFSCs upon cyclopamine ( Cyclo ) treatment , whereas dorsomorphin ( Dorso ) treatment leads to depletion of HFSCs ( mean ± SD ; n = 3 ; * p ≤ 0 . 05 , Kruskal - Wallis ) . D . Heatmap of significantly ( padj value < 0 . 05 ) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h . HFSC identity genes ( triangles ) and genes implicated in HFSC lineage progression ( circles ) are indicated . E . RT - qPCR analysis of 3C cultures treated as in ( D ) confirms that Id 1 , 2 and 3 are significantly upregulated in Cyclo treated cells ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U test ) . F . RT - qPCR analysis of 3C cultures treated as in ( A ) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . H . Quantification of experiments as in E ( mean ± SD ; n = 3 - 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S6 and Dataset EV5 .

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

Figure 6A-B. Inhibitor treatment scheme (A) and FACS plots (B) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin (mean ±SD; n=3). C. Quantification of experiments as in (B) shows enrichment of HFSCs upon cyclopamine (Cyclo) treatment, whereas dorsomorphin (Dorso) treatment leads to depletion of HFSCs (mean ±SD; n=3; *p ≤ 0.05, Kruskal-Wallis).D. Heatmap of significantly (padj value<0.05) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h. HFSC identity genes (triangles) and genes implicated in HFSC lineage progression (circles) are indicated.E. RT-qPCR analysis of 3C cultures treated as in (D) confirms that Id 1,2 and 3 are significantly upregulated in Cyclo treated cells (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).F. RT-qPCR analysis of 3C cultures treated as in (A) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).H. Quantification of experiments as in E (mean ±SD; n=3-4; * p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S6 and Dataset EV5.


Figure 1 . ( e ) Binding of monoclonal antibodies to 2019 - nCoV RBD determined by ELISA . ( f ) Binding profiles of 2019 - nCoV RBD to ACE2 and antibodies ( g ) competition of CR3022 and ACE2 with 2019 - nCoV RBD measured by BLI in OctetRED96 . Binding kinetics was evaluated using a 1 : 1 Langmuir binding model by ForteBio Data Analysis 7 . 0 software .

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Figure 1.(e) Binding of monoclonal antibodies to 2019-nCoV RBD determined by ELISA.(f) Binding profiles of 2019-nCoV RBD to ACE2 and antibodies(g) competition of CR3022 and ACE2 with 2019-nCoV RBD measured by BLI in OctetRED96. Binding kinetics was evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 7.0 software.


Figure 1B , Immunofluorescence staining of single nuclei for H3K79me3 or H3K79me2 ( green ) and the pericentromeric heterochromatin marker HMGA1 ( red ) . Upper panels , prometaphase ; lower panels , interphase . DNA is stained with DAPI ( blue ) . Scale bar , 2µm . H , H3K79me2 and H3K79me3 ChIP - seq enrichment relative to input at selected repetitive element sequences . K , Immunofluorescence staining for H3K79me3 in control and Dot1l KO nuclei . Scale bar , 2µm . M , DNA Fluorescence in situ hybridization ( DNA FISH ) for the mouse major satellite element with immunofluorescence staining for H3K79me3 ( top ) or H3K79me2 ( bottom ) in single mESC nuclei . Scale bar , 5µm . Plot shows Pearson ' s correlation values between FISH and H3K79me2 or H3K79me3 signal , each point representing an individual nucleus . * * * p < 0 . 0001 , two - tailed unpaired t - test .

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Figure 1B, Immunofluorescence staining of single nuclei for H3K79me3 or H3K79me2 (green) and the pericentromeric heterochromatin marker HMGA1 (red). Upper panels, prometaphase; lower panels, interphase. DNA is stained with DAPI (blue). Scale bar, 2µm.H, H3K79me2 and H3K79me3 ChIP-seq enrichment relative to input at selected repetitive element sequences.K, Immunofluorescence staining for H3K79me3 in control and Dot1l KO nuclei. Scale bar, 2µm.M, DNA Fluorescence in situ hybridization (DNA FISH) for the mouse major satellite element with immunofluorescence staining for H3K79me3 (top) or H3K79me2 (bottom) in single mESC nuclei. Scale bar, 5µm. Plot shows Pearson's correlation values between FISH and H3K79me2 or H3K79me3 signal, each point representing an individual nucleus. ***p<0.0001, two-tailed unpaired t-test.


Figure 2B , Metagene plots showing mean ChIP signal for H3K79me2 and H3K79me3 at downregulated and upregulated genes identified in ( A ) . F , RNA FISH for major satellite transcripts and simultaneous staining for H3K79me3 in control and Dot1l KO mESCs . Scale bar , 5µm . Below left , quantitation of fluorescence intensity along a line bisecting a representative nucleus . Bisecting lines used to generate the plots are shown in the merged images . Below right , quantitation of total FISH fluorescence signal for n = 46 cells of each genotype . Line summaries show mean ± standard deviation . * * * p < 0 . 0001 , unpaired Welch ' s t - test .

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Figure 2B, Metagene plots showing mean ChIP signal for H3K79me2 and H3K79me3 at downregulated and upregulated genes identified in (A).F, RNA FISH for major satellite transcripts and simultaneous staining for H3K79me3 in control and Dot1l KO mESCs. Scale bar, 5µm. Below left, quantitation of fluorescence intensity along a line bisecting a representative nucleus. Bisecting lines used to generate the plots are shown in the merged images. Below right, quantitation of total FISH fluorescence signal for n=46 cells of each genotype. Line summaries show mean ± standard deviation. ***p<0.0001, unpaired Welch's t-test.


Figure 3A , ChIP - qPCR for HP1β at selected repeat elements in Dot1l KO and wild type mESCs . Bars represent mean of n = 2 biological replicates . Neg . control indicates an intergenic locus ( mm10 chr19 : 51758385 - 58758494 ) not strongly associated with either euchromatin or heterochromatin . B , Immunofluorescence staining of HP1β ( green ) in wild type and Dot1l KO mESCs . DNA is stained with DAPI ( blue ) . Quantification of signal is shown at right . Each point represents one nucleus ( Dot1l KO n = 83 and control n = 104 nuclei ) . Scale bar , 10µm . * * * p < 0 . 0001 , Mann - Whitney U test . G , Western blot for co - immunoprecipitation of DOT1L and SMARCA5 in mESCs . AF10 is a positive control and GAPDH is a loading control . H , Immunofluorescence staining of SMARCA5 ( green ) and HMGA1 ( red ) in mESCs , indicating localization of SMARCA5 to heterochromatic regions . Arrowheads indicate an example of SMARCA5 enrichment at PCH . DNA is stained with DAPI ( blue ) . Scale bar , 4µm . Bottom , quantitation of fluorescence intensity in each channel along a line bisecting the nucleus . J , H3K79me3 enrichment at major satellite sequences in Smarca5 knockdown ( KD ) relative to scrambled shRNA control mESCs measured by ChIP - qPCR . Bars represent mean of n = 3 biological replicates and error bars represent ± SD . p = 0 . 0876 for the major satellite element ( one - sample t - test compared to expected fold change of 1 , shown as a dashed line ) . L , Immunofluorescence staining of H3K79me3 ( red ) in mouse 3T3 fibroblasts transduced with a vector expressing GFP and a short hairpin against Smarca5 ( shSmarca5 ) compared to untransduced control . Arrowheads indicate DAPI - dense chromocenters . Merge includes GFP channel for transduced cells . Scale bar , 10µm .

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

Figure 3A, ChIP-qPCR for HP1β at selected repeat elements in Dot1l KO and wild type mESCs. Bars represent mean of n=2 biological replicates. Neg. control indicates an intergenic locus (mm10 chr19:51758385-58758494) not strongly associated with either euchromatin or heterochromatin.B, Immunofluorescence staining of HP1β (green) in wild type and Dot1l KO mESCs. DNA is stained with DAPI (blue). Quantification of signal is shown at right. Each point represents one nucleus (Dot1l KO n=83 and control n=104 nuclei). Scale bar, 10µm. ***p<0.0001, Mann-Whitney U test.G, Western blot for co-immunoprecipitation of DOT1L and SMARCA5 in mESCs. AF10 is a positive control and GAPDH is a loading control.H, Immunofluorescence staining of SMARCA5 (green) and HMGA1 (red) in mESCs, indicating localization of SMARCA5 to heterochromatic regions. Arrowheads indicate an example of SMARCA5 enrichment at PCH. DNA is stained with DAPI (blue). Scale bar, 4µm. Bottom, quantitation of fluorescence intensity in each channel along a line bisecting the nucleus.J, H3K79me3 enrichment at major satellite sequences in Smarca5 knockdown (KD) relative to scrambled shRNA control mESCs measured by ChIP-qPCR. Bars represent mean of n=3 biological replicates and error bars represent ± SD. p=0.0876 for the major satellite element (one-sample t-test compared to expected fold change of 1, shown as a dashed line).L, Immunofluorescence staining of H3K79me3 (red) in mouse 3T3 fibroblasts transduced with a vector expressing GFP and a short hairpin against Smarca5 (shSmarca5) compared to untransduced control. Arrowheads indicate DAPI-dense chromocenters. Merge includes GFP channel for transduced cells. Scale bar, 10µm.


Figure 4A , Immunofluorescence staining of H3K79me3 ( green ) and H3S10P ( red ) in a single nucleus of control and Dot1l KO mESCs . DNA is stained with DAPI ( blue ) . Scale bar , 5 µm . B , Example metaphase spreads in wild type and Dot1l KO mESCs showing examples of a chromosome break , premature centromere separation , and chromosome fusion ( yellow boxes ) . Scale bar , 5 µm . F , Western blot for the DNA damage marker γH2A . X in control and Dot1l KO mESCs . Quantitation of signal on the blot for n = 2 biological replicates is shown .

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Figure 4A, Immunofluorescence staining of H3K79me3 (green) and H3S10P (red) in a single nucleus of control and Dot1l KO mESCs. DNA is stained with DAPI (blue). Scale bar, 5 µm.B, Example metaphase spreads in wild type and Dot1l KO mESCs showing examples of a chromosome break, premature centromere separation, and chromosome fusion (yellow boxes). Scale bar, 5 µm.F, Western blot for the DNA damage marker γH2A.X in control and Dot1l KO mESCs. Quantitation of signal on the blot for n=2 biological replicates is shown.


Figure 5A , Immunofluorescence staining of H3K79me3 ( green ) and H3K9me3 ( red ) in wild type mouse embryos during the first 48 hours of preimplantation development . Bottom images show an embryo treated with DOT1L inhibitor at 48 hours post fertilization ( hpf ) . DNA is stained with DAPI ( blue ) . Scale bars , 10 µm ( main image ) or 4 µm ( inset ) . Quantitation of pixel - by - pixel fluorescence intensity for H3K9me3 and H3K79me3 for a representative embryo is shown at right . AU , arbitrary units . B , Quantitation of numbers of total , H3K9me3 - positive , and H3K79me3 - positive chromocenters in control and inhibitor - treated embryos at 48 hpf . Chromocenters were defined as DAPI - dense foci . Each point represents one nucleus . N = 69 ( control ) or n = 20 nuclei ( inhibitor treated ) in n = 19 ( control ) or n = 5 ( inhibitor treated ) embryos * p < 0 . 05 , * * * p < 0 . 001 , two - tailed unpaired t - test .

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

Figure 5A, Immunofluorescence staining of H3K79me3 (green) and H3K9me3 (red) in wild type mouse embryos during the first 48 hours of preimplantation development. Bottom images show an embryo treated with DOT1L inhibitor at 48 hours post fertilization (hpf). DNA is stained with DAPI (blue). Scale bars, 10 µm (main image) or 4 µm (inset). Quantitation of pixel-by-pixel fluorescence intensity for H3K9me3 and H3K79me3 for a representative embryo is shown at right. AU, arbitrary units.B, Quantitation of numbers of total, H3K9me3-positive, and H3K79me3-positive chromocenters in control and inhibitor-treated embryos at 48 hpf. Chromocenters were defined as DAPI-dense foci. Each point represents one nucleus. N=69 (control) or n=20 nuclei (inhibitor treated) in n=19 (control) or n=5 (inhibitor treated) embryos *p<0.05, ***p<0.001, two-tailed unpaired t-test.


Figure 6A , Volcano plot showing differentially expressed single - copy genes in DOT1L inhibitor - treated vs control embryos at 56hpf ( 8 cell stage ) . Each dot signifies one gene . Differentially expressed genes ( adjusted p - value ≤ 0 . 05 ; no threshold for log2 fold change ) are shown in red . P - values were corrected for multiple hypothesis testing using the Benjamini - Hochberg method .

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Figure 6A, Volcano plot showing differentially expressed single-copy genes in DOT1L inhibitor-treated vs control embryos at 56hpf (8 cell stage). Each dot signifies one gene. Differentially expressed genes (adjusted p-value ≤ 0.05; no threshold for log2 fold change) are shown in red. P-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method.


Figure 1Antibacterial competition assay . E . coli K12 recipient cells ( W3110 gfp + , kanR ) were mixed with the indicated EAEC attacker cells ( 1 : 4 ratio ) : WT , ∆ T6SS - 1 and ∆ tle1 - tli1 carrying pBAD18 and pBAD33 vectors , or producing the indicated proteins , and spotted on SIM 0 . 02 % arabinose agar plates for 4 - h at 37 ° C . The image of a representative bacterial spot is shown . The number of recovered prey cells is indicated in the upper graph ( in log of colony - forming units ( cfu ) ) . The black , dark grey and light grey circles indicate values obtained from three different spots , and the average is indicated by the bar . Western blot analysis of the production of Tle1V and Tle1 ∆ 1 - 26 V is shown in the inset . The experiment was performed in triplicate and a representative result is shown . Co - immunoprecipitation assay . Lysates from E . coli K - 12 W3110 cells producing VSVG - tagged Tle1 ( Tle1V ) or VSVG - tagged Tle1 ∆ 1 - 26 truncated variant ( Tle1 ∆ 1 - 26 V ) were mixed with lysates from W3110 cells producing FLAG - tagged VgrG ( VgrGFL ) or not ( - , empty vector ) and subjected to immunoprecipitation on anti - FLAG - coupled beads . The mixed soluble lysates ( Input ) and the immunoprecipitated material ( IP ) were subjected to 12 . 5 % SDS - PAGE and immunodetected with anti - FLAG ( upper panel ) and anti - VSVG ( lower panel ) monoclonal antibodies . Molecular weight markers ( in kDa ) are indicated on the left . This experiment was performed in triplicate and a representative result is shown .

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Figure 1Antibacterial competition assay. E. coli K12 recipient cells (W3110 gfp+, kanR) were mixed with the indicated EAEC attacker cells (1:4 ratio): WT, ∆T6SS-1 and ∆tle1-tli1 carrying pBAD18 and pBAD33 vectors, or producing the indicated proteins, and spotted on SIM 0.02 % arabinose agar plates for 4-h at 37°C. The image of a representative bacterial spot is shown. The number of recovered prey cells is indicated in the upper graph (in log of colony-forming units (cfu)). The black, dark grey and light grey circles indicate values obtained from three different spots, and the average is indicated by the bar. Western blot analysis of the production of Tle1V and Tle1∆1-26 V is shown in the inset. The experiment was performed in triplicate and a representative result is shown.Co-immunoprecipitation assay. Lysates from E. coli K-12 W3110 cells producing VSVG-tagged Tle1 (Tle1V) or VSVG-tagged Tle1∆1-26 truncated variant (Tle1∆1-26 V) were mixed with lysates from W3110 cells producing FLAG-tagged VgrG (VgrGFL) or not (-, empty vector) and subjected to immunoprecipitation on anti-FLAG-coupled beads. The mixed soluble lysates (Input) and the immunoprecipitated material (IP) were subjected to 12.5% SDS-PAGE and immunodetected with anti-FLAG (upper panel) and anti-VSVG (lower panel) monoclonal antibodies. Molecular weight markers (in kDa) are indicated on the left. This experiment was performed in triplicate and a representative result is shown.


Figure 5Specific phospholipase A1 ( PLA1 ) activity measurements of Tle1H , SVgrG - Tle1H complex , and isolated SVgrG . Thermomyces lanuginosus lipase ( TLL ) was used as positive standard for PLA1 activity . Mean values and standard deviation from at least three independent assays are shown . Statistical analysis relative to the Tle1 activity is indicated . * * * P < 0 . 001 ( P = 0 , 0001998 ) , unpaired two - sample Fisher - Pitman permutation test . VgrG inhibition of Tle1H . Specific phospholipase A1 ( PLA1 ) activity measurements of purified Tle1H in the presence of 1 : 10 molar ratio of lysozyme from chicken egg white ( Lyso ) and increasing molar ratio ( 0 , 1 , 5 , and 10 ) of SVgrG or SVgrG1 - 490 . Inhibition experiments with SVgrG were repeated three times with independent protein preparations , each measured in triplicate . Inhibition experiments with SVgrG1 - 490 were repeated twice with two independent protein preparations , each measured in triplicate . Mean values and standard deviation are shown . Statistical analysis relative to the Tle1 activity is indicated . ns , non - significant ( P > 0 . 05 ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unpaired two - sample Fisher - Pitman permutation test . ( P values : Lyso , 0 . 7006 ; VgrG 1 : 1 , 0 . 004946 ; VgrG 1 : 5 , 1 . 448e - 07 ; VgrG1 : 10 , 1 . 448e - 07 ; VgrG1 - 490 1 : 1 , 0 . 5627 ; VgrG1 - 490 1 : 5 , 0 . 2977 ; VgrG1 - 490 1 : 10 , 0 . 002631 ) . Representative SDS - PAGE / Coomassie blue staining of the Tle1H , SVgrG and SVgrG1 - 490 purified proteins used in B ( 5 μg / well ) .

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Figure 5Specific phospholipase A1 (PLA1) activity measurements of Tle1H, SVgrG-Tle1H complex, and isolated SVgrG. Thermomyces lanuginosus lipase (TLL) was used as positive standard for PLA1 activity. Mean values and standard deviation from at least three independent assays are shown. Statistical analysis relative to the Tle1 activity is indicated. ***P<0.001 (P=0,0001998), unpaired two-sample Fisher-Pitman permutation test.VgrG inhibition of Tle1H. Specific phospholipase A1 (PLA1) activity measurements of purified Tle1H in the presence of 1:10 molar ratio of lysozyme from chicken egg white (Lyso) and increasing molar ratio (0, 1, 5, and 10) of SVgrG or SVgrG1-490. Inhibition experiments with SVgrG were repeated three times with independent protein preparations, each measured in triplicate. Inhibition experiments with SVgrG1-490 were repeated twice with two independent protein preparations, each measured in triplicate. Mean values and standard deviation are shown. Statistical analysis relative to the Tle1 activity is indicated. ns, non-significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, unpaired two-sample Fisher-Pitman permutation test. (P values: Lyso, 0.7006; VgrG 1:1, 0.004946; VgrG 1:5, 1.448e-07; VgrG1:10, 1.448e-07; VgrG1-490 1:1, 0.5627; VgrG1-490 1:5, 0.2977; VgrG1-490 1:10, 0.002631).Representative SDS-PAGE / Coomassie blue staining of the Tle1H, SVgrG and SVgrG1-490 purified proteins used in B (5 μg/well).


Figure 1 ( A , B ) PTPRN2 ( A ) and PLCB1 ( B ) expression levels were determined by qRT - PCR . ( C ) Bioluminescence imaging quantification of lung colonization by 40 , 000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin . For shCntrl , sh1PTPRN2 , N = 5 mice / group . For sh2PTPRN2 N = 6 mice . Right , H & amp ; amp ; E staining of representative lung sections . ( D ) Bioluminescence imaging quantification of lung colonization by 40 , 000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA . For siCntrl , N = 5 mice . For si1PLCβ1 , si2PLCβ1 , N = 6 mice / group . Right , H & amp ; amp ; E staining of representative lung sections . ( E , F ) PTPRN2 ( E ) and PLCB1 ( F ) levels were analyzed in human breast cancers ( stages I - IV ) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III ( Origene , N = 97 ) . Expression levels were normalized to levels in normal tissue for each gene . ( G ) Kaplan - Meier curve representing overall survival of a cohort of breast cancer patients ( N = 528 ) as a function of their primary tumor ' s PTPRN2 and PLCB1 expression levels ( data from the TCGA Research Network ( Cancer Genome Atlas , 2012 ) ) . Patients whose primary tumors ' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low ( blue ) or high ( red ) expression . ( H ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer patients ( N = 1609 ) as a function of their primary tumor ' s PTPRN2 and PLCB1 expression levels ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . Patients ' primary tumors ' PTPRN2 and PLCB1 expression levels were classified as low ( blue ) or high ( red ) expression .

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 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 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 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 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 1(A,B) PTPRN2 (A) and PLCB1 (B) expression levels were determined by qRT-PCR.(C) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 breast cancer cells transduced with shRNAs targeting PTPRN2 or a control hairpin. For shCntrl, sh1PTPRN2, N = 5 mice/group. For sh2PTPRN2 N = 6 mice. Right, H&amp;E staining of representative lung sections.(D) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells transfected with siRNA targeting PLCβ1 or a control siRNA. For siCntrl, N = 5 mice. For si1PLCβ1, si2PLCβ1, N = 6 mice/group. Right, H&amp;E staining of representative lung sections.(E,F) PTPRN2 (E) and PLCB1 (F) levels were analyzed in human breast cancers (stages I-IV) and normal breast tissue from TissueScan qPCR Array Breast Cancer Panels II and III (Origene, N = 97). Expression levels were normalized to levels in normal tissue for each gene.(G) Kaplan-Meier curve representing overall survival of a cohort of breast cancer patients (N = 528) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (data from the TCGA Research Network (Cancer Genome Atlas, 2012)). Patients whose primary tumors' PTPRN2 and PLCB1 expression levels were higher or lower than the median of the population were classified as low (blue) or high (red) expression.(H) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's PTPRN2 and PLCB1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' PTPRN2 and PLCB1 expression levels were classified as low (blue) or high (red) expression.


Figure 2 ( A ) Matrigel invasion by 50 , 000 LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group . ( B ) Migration assay by 100 , 000 LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . Data normalized to control values . N = 6 inserts / group . Right , representative images of the migration assay . Scale bar , 100 μm . ( C ) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA . N = 5 wells / group . Right , representative images of the scratch assay . ( D , E ) MDA - MB - 231 cells transduced with PTPRN2 , PTPRN2C945A , or control vector were subjected to the Matrigel invasion ( D ) and migration assays ( E ) . N = 5 inserts / group . ( F ) Bioluminescence imaging quantification of lung colonization by 40 , 000 MDA - MB - 231 cells overexpressing PTPRN2 , PTPRN2C945A , or control vector . N = 5 mice / group . Right , H & amp ; amp ; E staining of representative lung sections . ( G , H ) MDA - MB - 231 cells transduced with PLCβ1 , PLCβ1H331Q , or control vector were subjected to the Matrigel invasion ( G ) and migration assays ( H ) . N = 5 inserts / group . ( I ) Bioluminescence imaging quantification of lung colonization by 40 , 000 MDA - MB - 231 cells overexpressing PLCβ1 , PLCβ1H331Q , or control vector . For Cntrl and PLCβ1H331Q OE , N = 6 mice / group . For PLCβ1 OE , N = 5 mice . Right , H & amp ; amp ; E staining of representative lung sections .

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

Figure 2(A) Matrigel invasion by 50,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group.(B) Migration assay by 100,000 LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. Data normalized to control values. N = 6 inserts/group. Right, representative images of the migration assay. Scale bar, 100 μm.(C) Quantification of area covered by cells 24 h after a scratch was made through confluent cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA. N = 5 wells/group. Right, representative images of the scratch assay.(D,E) MDA-MB-231 cells transduced with PTPRN2, PTPRN2C945A, or control vector were subjected to the Matrigel invasion (D) and migration assays (E). N = 5 inserts/group.(F) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PTPRN2, PTPRN2C945A, or control vector. N = 5 mice/group. Right, H&amp;E staining of representative lung sections.(G,H) MDA-MB-231 cells transduced with PLCβ1, PLCβ1H331Q, or control vector were subjected to the Matrigel invasion (G) and migration assays (H). N = 5 inserts/group.(I) Bioluminescence imaging quantification of lung colonization by 40,000 MDA-MB-231 cells overexpressing PLCβ1, PLCβ1H331Q, or control vector. For Cntrl and PLCβ1H331Q OE, N = 6 mice/group. For PLCβ1 OE, N = 5 mice. Right, H&amp;E staining of representative lung sections.


Figure 3 ( A ) Mean fluorescence intensity of membrane PI ( 4 , 5 ) P2 was analyzed in MDA - MB - 231 and LM2 cells immunostained with PI ( 4 , 5 ) P2 antibody using fluorescence microscopy . N = 50 cells / group . Scale bar , 10 μm . ( B ) Bioluminescence imaging quantification of lung colonization by 40 , 000 LM2 cells treated with carrier incubated with PI ( 4 , 5 ) P2 or carrier alone for one hour and then immediately injected . Right , H & amp ; amp ; E staining of representative lung sections . N = 6 mice / group . ( C , D ) MDA - MB - 231 cells overexpressing PTPRN2 , PLCβ1 , or a control vector ( C ) or LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or a control siRNA ( D ) were immunostained for PI ( 4 , 5 ) P2 levels and analyzed by fluorescence microscopy . Mean fluorescence intensity of plasma membrane levels of the lipid were quantified . N = 50 cells / group . Left , representative immunofluorescence images of cells stained with anti - PI ( 4 , 5 ) P2 antibody ( red ) and 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Scale bar , 10 μm .

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

Figure 3(A) Mean fluorescence intensity of membrane PI(4,5)P2 was analyzed in MDA-MB-231 and LM2 cells immunostained with PI(4,5)P2 antibody using fluorescence microscopy. N = 50 cells/group. Scale bar, 10 μm.(B) Bioluminescence imaging quantification of lung colonization by 40,000 LM2 cells treated with carrier incubated with PI(4,5)P2 or carrier alone for one hour and then immediately injected. Right, H&amp;E staining of representative lung sections. N=6 mice/group.(C,D) MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or a control vector (C) or LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or a control siRNA (D) were immunostained for PI(4,5)P2 levels and analyzed by fluorescence microscopy. Mean fluorescence intensity of plasma membrane levels of the lipid were quantified. N = 50 cells/group. Left, representative immunofluorescence images of cells stained with anti-PI(4,5)P2 antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 10 μm.


Figure 4 ( A , B ) LM2 cells transfected with siRNA targeting PTPRN2 ( A ) , PLCβ1 ( B ) or a control siRNA were transfected with Lyn11 - FRB and INPP5E - FKBP , treated with either DMSO or 100 nm rapamycin and subjected to the migration assay . N = 5 inserts / group . ( C , D ) MDA - MB - 231 cells overexpressing PTPRN2 ( C ) , PLCβ1 ( D ) or control vector were treated with carrier alone or carrier incubated with PI ( 4 , 5 ) P2 for one hour and then immediately subjected to the migration assay . N = 5 inserts / group . ( C , D ) MDA - MB - 231 cells overexpressing PTPRN2 ( C ) , PLCβ1 ( D ) or control vector were treated with carrier alone or carrier incubated with PI ( 4 , 5 ) P2 for one hour and then immediately subjected to the migration assay . N = 5 inserts / group . ( E ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer patients ( N = 1609 ) as a function of their primary tumor ' s mean PIP5K1A , PIP5KB , and PIP5KC expression levels ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . Patients ' primary tumors ' combined PIP5K expression levels were classified as low ( blue ) or high ( red ) expression . ( F , G ) Migration ( F ) and Matrigel invasion ( G ) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector . Data normalized to control values . N = 5 inserts / group .

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

Figure 4(A,B) LM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11-FRB and INPP5E-FKBP, treated with either DMSO or 100 nm rapamycin and subjected to the migration assay. N = 5 inserts/group.(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.(C,D) MDA-MB-231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for one hour and then immediately subjected to the migration assay. N = 5 inserts/group.(E) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's mean PIP5K1A, PIP5KB, and PIP5KC expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' combined PIP5K expression levels were classified as low (blue) or high (red) expression.(F,G) Migration (F) and Matrigel invasion (G) of LM2 cells transduced with a retroviral vector overexpressing PIP5K1A or control vector. Data normalized to control values. N = 5 inserts/group.


Figure 5 ( A ) Kaplan - Meier curve representing distal metastasis - free survival of a cohort of breast cancer patients ( N = 1609 ) as a function of their primary tumor ' s CFL1 expression levels ( Data from KMPlot ( Gyorffy et al . , 2010 ) ) . Patients ' primary tumors ' CFL1 expression levels were classified as low ( blue ) or high ( red ) expression . ( B , C ) Membrane and membrane - associated proteins were purified from cells transfected with siRNA targeting PTPRN2 ( B ) or PLCβ1 ( C ) or control siRNA . Fractions were subjected to Western blot analysis for CFL1 and EGFR levels . Right , densitometry analysis of CFL1 levels normalized to EGFR levels . ( B , C ) Membrane and membrane - associated proteins were purified from cells transfected with siRNA targeting PTPRN2 ( B ) or PLCβ1 ( C ) or control siRNA . Fractions were subjected to Western blot analysis for CFL1 and EGFR levels . Right , densitometry analysis of CFL1 levels normalized to EGFR levels . ( D , E ) Membrane and membrane - associated proteins were purified from cells overexpressing PTPRN2 ( D ) , PLCβ1 ( E ) or a control vector . Fractions were subjected to Western blot analysis for CFL1 and EGFR levels . Right , densitometry analysis of CFL1 levels normalized to EGFR levels . ( D , E ) Membrane and membrane - associated proteins were purified from cells overexpressing PTPRN2 ( D ) , PLCβ1 ( E ) or a control vector . Fractions were subjected to Western blot analysis for CFL1 and EGFR levels . Right , densitometry analysis of CFL1 levels normalized to EGFR levels . ( F ) LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were partially permeabilized and incubated with biotin - actin monomers . Cells were stained for incorporated biotin - actin monomers using Streptavidin - 555 ( red ) and DAPI ( blue ) . Right , quantification of mean fluorescence intensity of incorporated biotin - actin monomers . N = 100 cells / group . Scale bar , 20 μm .

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

Figure 5(A) Kaplan-Meier curve representing distal metastasis-free survival of a cohort of breast cancer patients (N = 1609) as a function of their primary tumor's CFL1 expression levels (Data from KMPlot (Gyorffy et al., 2010)). Patients' primary tumors' CFL1 expression levels were classified as low (blue) or high (red) expression.(B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(B,C) Membrane and membrane-associated proteins were purified from cells transfected with siRNA targeting PTPRN2 (B) or PLCβ1 (C) or control siRNA. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(D,E) Membrane and membrane-associated proteins were purified from cells overexpressing PTPRN2 (D), PLCβ1 (E) or a control vector. Fractions were subjected to Western blot analysis for CFL1 and EGFR levels. Right, densitometry analysis of CFL1 levels normalized to EGFR levels.(F) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were partially permeabilized and incubated with biotin-actin monomers. Cells were stained for incorporated biotin-actin monomers using Streptavidin-555 (red) and DAPI (blue). Right, quantification of mean fluorescence intensity of incorporated biotin-actin monomers. N = 100 cells/group. Scale bar, 20 μm.


Figure 6 ( A ) LM2 cells transfected with siRNA targeting PTPRN2 , PLCβ1 , or control siRNA were stained with phalloidin ( red ) and DAPI ( blue ) and analyzed using fluorescence microscopy . Right , mean fluorescence intensity quantification of whole cell phalloidin signal . N = 40 cells / group . Scale bar , 10 μm . ( B ) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA - MB - 231 cells overexpressing PTPRN2 , PLCβ1 , or control vector . N = 40 cells / group . ( C , D ) MDA - MB - 231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration ( C ) or invasion ( D ) assays . N = 5 inserts / group . ( E ) MDA - MB - 231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay . N = 5 inserts / group . ( F ) Bioluminescence imaging quantification of 40 , 000 MDA - MB - 231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1 . Right , H & amp ; amp ; E staining of representative lung sections . N = 5 mice / group . ( G , H ) MDA - MB - 231 cells overexpressing PTPRN2 ( G ) or PLCβ1 ( H ) were transfected with siRNA targeting the 3 ' UTR of CFL1 to deplete endogenous CFL1 . Cells were further transfected with plasmids encoding either GFP - CFL1 - WT or GFP - CFL1 - Lck and subjected to the migration assay . N = 5 inserts / group .

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

Figure 6(A) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were stained with phalloidin (red) and DAPI (blue) and analyzed using fluorescence microscopy. Right, mean fluorescence intensity quantification of whole cell phalloidin signal. N = 40 cells/group. Scale bar, 10 μm.(B) Mean fluorescence intensity quantification of whole cell phalloidin signal in MDA-MB-231 cells overexpressing PTPRN2, PLCβ1, or control vector. N = 40 cells/group.(C,D) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PTPRN2 overexpression and subjected to the migration (C) or invasion (D) assays. N = 5 inserts/group.(E) MDA-MB-231 cells were transfected with siRNAs targeting CFL1 or a control siRNA in the setting of control or PLCβ1 overexpression were subjected to the migration assay. N = 5 inserts/group.(F) Bioluminescence imaging quantification of 40,000 MDA-MB-231 cells overexpressing PTPRN2 or control vector and transfected with control siRNA or siRNA targeting CFL1. Right, H&amp;E staining of representative lung sections. N = 5 mice/group.(G,H) MDA-MB-231 cells overexpressing PTPRN2 (G) or PLCβ1 (H) were transfected with siRNA targeting the 3'UTR of CFL1 to deplete endogenous CFL1. Cells were further transfected with plasmids encoding either GFP-CFL1-WT or GFP-CFL1-Lck and subjected to the migration assay. N = 5 inserts/group.


Figure 2 ( A , B ) ELISA screen of the lectin - Fc library against full - length trimeric SARS - CoV - 2 Spike ( A ) or monomeric RBD ( B ) . Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value . Lectin - Fc fusion proteins with a normalized OD > 0 . 5 in either ( A ) or ( B ) are indicated in both panels . ( C ) Lectin - Fc and human ACE2 - mIgG1 Fc - fusion protein ( hACE2 ) binding to untreated or heat - denatured full - length SARS - CoV - 2 Spike by ELISA . hACE2 - mIgG1 was used as control for complete denaturation of Spike protein . Results are shown as mean OD values ± SD normalized to the BSA control ( technical replicates , N = 3 ) . ( D ) Lectin - Fc binding to full - length SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with the Spike protein . Results are shown as mean OD values ± SD normalized to BSA controls ( technical replicates , N = 3 ) .

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 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

Figure 2(A,B) ELISA screen of the lectin-Fc library against full-length trimeric SARS-CoV-2 Spike (A) or monomeric RBD (B). Results are shown as mean OD values of 2 replicates normalized against a BSA control and ranked by value. Lectin-Fc fusion proteins with a normalized OD > 0.5 in either (A) or (B) are indicated in both panels.(C) Lectin-Fc and human ACE2-mIgG1 Fc-fusion protein (hACE2) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike by ELISA. hACE2-mIgG1 was used as control for complete denaturation of Spike protein. Results are shown as mean OD values ± SD normalized to the BSA control (technical replicates, N=3).(D) Lectin-Fc binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized to BSA controls (technical replicates, N=3).


Figure 3 ( E ) High speed AFM of single trimeric Spike visualizing the real - time interaction dynamics with lectins . Top panel shows 5 frames of trimeric Spike alone imaged on mica . Middle and bottom panels show 5 sequential frames of trimeric Spike / Clec4g and trimeric Spike / CD209c complexes , acquired at a rate of 153 . 6 and 303 ms / frame , respectively . Association and dissociation events between lectin and Spike are indicated by white and red arrows , respectively . The blue dotted ellipses display the core of the complexes showing low conformational mobility . Color schemes indicate height of the molecules in nanometers ( nm ) . Volumes of single trimeric Spike , trimeric Spike / Clec4g and trimeric Spike / CD209c complexes are indicated , as well as numbers of lectins bound to trimeric Spike , averaged over the experimental recording period .

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

Figure 3(E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top panel shows 5 frames of trimeric Spike alone imaged on mica. Middle and bottom panels show 5 sequential frames of trimeric Spike/Clec4g and trimeric Spike/CD209c complexes, acquired at a rate of 153.6 and 303 ms/frame, respectively. Association and dissociation events between lectin and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike, trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.


Figure 4 ( A ) ELISA analyses of human lectin - hIgG1 Fc - fusion protein ( hCLEC4g , hCD209 , hCD299 ) binding to untreated or heat - denatured full - length SARS - CoV - 2 Spike . A human ACE2 - hIgG1 - Fc fusion protein ( hACE2 ) was used to control for the complete denaturation of Spike . Results are shown as mean OD values ± SD normalized to a BSA control ( technical replicates , N = 3 ) . ( B ) hCLEC4g , hCD209 , and hCD299 binding to full - length SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with the Spike protein . Results are shown as mean OD values ± SD normalized against the BSA control ( technical replicates , N = 3 ) . ( C , D ) SPR analysis with immobilized full - length trimeric Spike , probed with various concentrations of hCLEC4G ( C ) and hCD209 ( D ) .

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

Figure 4(A) ELISA analyses of human lectin-hIgG1 Fc-fusion protein (hCLEC4g, hCD209, hCD299) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike. A human ACE2-hIgG1-Fc fusion protein (hACE2) was used to control for the complete denaturation of Spike. Results are shown as mean OD values ± SD normalized to a BSA control (technical replicates, N=3).(B) hCLEC4g, hCD209, and hCD299 binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized against the BSA control (technical replicates, N=3).(C, D) SPR analysis with immobilized full-length trimeric Spike, probed with various concentrations of hCLEC4G (C) and hCD209 (D).


Figure 6 ( C ) Infectivity of mouse Clec4g , CD209c , Asgr1 and hCLEC4G pre - treated SARS - CoV - 2 virus in Vero E6 cells . Data represent two to three independent experiments for Clec4g , CD209c and hCLEC4G lectins ( N = 5 - 9 ) and , one experiment for Asgr1 ( N = 2 - 4 ) . ( D ) Infectivity of mouse Clec4g , CD209c , Asgr1 and hCLEC4G pre - treated SARS - CoV - 2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non - treated SARS - CoV - 2 infected Calu3 cells measured in quadruplicates ( technical replicates ) . : ( C ) and ( D ) Viral RNA was measured with qRT - PCR 15 hours after infection with 0 . 02 MOI ( 103 PFUs ) of SARS - CoV - 2 . Data are presented as fold changes of viral loads over mock ( only SARS - CoV - 2 was added ) treated controls ( mean log10 values ± SD ) . All no virus controls were negative for SARS - CoV - 2 .

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

Figure 6(C) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in Vero E6 cells. Data represent two to three independent experiments for Clec4g, CD209c and hCLEC4G lectins (N=5-9) and, one experiment for Asgr1 (N=2-4). (D) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non-treated SARS-CoV-2 infected Calu3 cells measured in quadruplicates (technical replicates). : (C) and (D) Viral RNA was measured with qRT-PCR 15 hours after infection with 0.02 MOI (103 PFUs) of SARS-CoV-2. Data are presented as fold changes of viral loads over mock (only SARS-CoV-2 was added) treated controls (mean log10 values ± SD). All no virus controls were negative for SARS-CoV-2.


Figure 1Survival curve of N . furzeri in the FLI facility . Recording of deaths starts at age of 5 wph , which corresponds to sexual maturity , and the colored dashed lines indicate the three age groups analysed in this study ( 5 animals / group ) , namely 5 weeks post hatching ( wph , young , sexual maturity ) , 12 wph ( adult ) and 39 wph ( old , past median lifespan ) of a wild - derived strain that exhibits a median lifespan of 7 - 8 months . Global protein - transcript correlation for each sample , grouped by age . RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA - seq and TMT - based proteomics data obtained from the same animal . An ANOVA test was performed to evaluate significance among the age groups ( mean correlation at 5 wph : 0 . 48 ; at 12 wph : 0 . 43 ; and at 39 wph : 0 . 33 ; p = 3 . 05e - 07 , n = 5 per age group ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range . Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels ( adj . p < 0 . 05 ) . The color gradients indicate gene density in the regions where individual points overlap . Numbers of genes in each quadrant and the value of Pearson ' s coefficient of correlation , r , are reported for each graph . Solid lines represent a spline fit ( r = 0 . 505 for genes significantly affected at both transcript and protein levels , p < 2 . 2 x 10 - 16 , D ) ( r = 0 . 126 , p = 0 . 007 , E ) . Mechanisms affecting protein abundance during aging . Bar plots are based on all the proteins affected in either one of the age comparisons ( adj . p < 0 . 05 ) . Proteins were divided in the following five groups : ( i ) proteins and transcripts with significant and consistent changes ( dark brown ) , ( ii ) proteins with significant changes , and with consistent changes of the transcripts ( light brown ) , ( iii ) proteins with no transcripts detected ( dark gray ) , ( iv ) proteins with transcripts whose translation is potentially regulated by miRNAs ( light gray ) , as assessed by the workflow displayed in ( Figure EV2D ) , ( v ) all the remaining proteins that we classified as regulated by other post - transcriptional mechanisms ( violet ) . pgs = protein groups .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Survival curve of N. furzeri in the FLI facility. Recording of deaths starts at age of 5 wph, which corresponds to sexual maturity, and the colored dashed lines indicate the three age groups analysed in this study (5 animals/group), namely 5 weeks post hatching (wph, young, sexual maturity), 12 wph (adult) and 39 wph (old, past median lifespan) of a wild-derived strain that exhibits a median lifespan of 7-8 months.Global protein-transcript correlation for each sample, grouped by age. RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA-seq and TMT-based proteomics data obtained from the same animal. An ANOVA test was performed to evaluate significance among the age groups (mean correlation at 5 wph: 0.48; at 12 wph: 0.43; and at 39 wph: 0.33; p = 3.05e-07, n=5 per age group). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels (adj. p < 0.05). The color gradients indicate gene density in the regions where individual points overlap. Numbers of genes in each quadrant and the value of Pearson's coefficient of correlation, r, are reported for each graph. Solid lines represent a spline fit (r = 0.505 for genes significantly affected at both transcript and protein levels, p < 2.2 x 10-16, D) (r = 0.126, p = 0.007, E).Mechanisms affecting protein abundance during aging. Bar plots are based on all the proteins affected in either one of the age comparisons (adj. p < 0.05). Proteins were divided in the following five groups: (i) proteins and transcripts with significant and consistent changes (dark brown), (ii) proteins with significant changes, and with consistent changes of the transcripts (light brown), (iii) proteins with no transcripts detected (dark gray), (iv) proteins with transcripts whose translation is potentially regulated by miRNAs (light gray), as assessed by the workflow displayed in (Figure EV2D), (v) all the remaining proteins that we classified as regulated by other post-transcriptional mechanisms (violet). pgs = protein groups.


Figure 2Abundance changes of ribosomal proteins and their transcripts during aging . Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots . Transcripts are displayed as light blue and proteins as dark blue boxes . Changes of individual proteins are displayed as dots ; orange dots identify significant cases ( adj . p < 0 . 05 , n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range . Brains from young ( 5 wph ) and old ( 39 wph ) were homogenized and clarified lysates separated by size - exclusion chromatography ( SEC ) . For each age group , two pools of brains were processed separately . For each experiment ( 4 in total ) , 39 fractions were collected along the chromatogram , digested into peptides and analysed by Data Independent Acquisition ( DIA ) quantitative mass spectrometry . Co - elution profiles for selected protein complexes . For each complex , the median abundance of all the quantified subunits was used to generate the complex profile across fractions ( Dataset EV5 ) . All the complex profiles are scaled to the max value ( set to 1 ) to make profiles comparable across experiments . The estimated molecular weight of the displayed complexes is indicated in brackets . Statistics of protein complexes undergoing stoichiometry changes with aging . Only protein complexes that had at least 5 members quantified were considered for each comparison . Complexes were considered affected if at least two members showed significant stoichiometry change ( adj . p < 0 . 05 and absolute log2 fold change > 0 . 5 ) . The complete list of stoichiometry changes is available in Dataset EV6 . Violin plots depicting interquartile ranges ( IQRs ) of individual members of protein complexes during aging . The IQR for each protein complex considered in G was calculated using transcript ( total RNA dataset , light blue ) or protein ( dark blue ) log2 fold changes between two age groups ( n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . * p < 0 . 05 , Wilcoxon Rank Sum test . The central line of the violin plots indicates the median value . Heat map showing relative protein fold changes for members of selected complexes affected by aging . Names of significantly affected members in the 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 and absolute log2 fold change > 0 . 5 ) are highlighted in bold with a star . Related to Figure EV3 and Dataset EV5 and EV6 .

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

Figure 2Abundance changes of ribosomal proteins and their transcripts during aging. Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots; orange dots identify significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.Brains from young (5 wph) and old (39 wph) were homogenized and clarified lysates separated by size-exclusion chromatography (SEC). For each age group, two pools of brains were processed separately. For each experiment (4 in total), 39 fractions were collected along the chromatogram, digested into peptides and analysed by Data Independent Acquisition (DIA) quantitative mass spectrometry.Co-elution profiles for selected protein complexes. For each complex, the median abundance of all the quantified subunits was used to generate the complex profile across fractions (Dataset EV5). All the complex profiles are scaled to the max value (set to 1) to make profiles comparable across experiments. The estimated molecular weight of the displayed complexes is indicated in brackets.Statistics of protein complexes undergoing stoichiometry changes with aging. Only protein complexes that had at least 5 members quantified were considered for each comparison. Complexes were considered affected if at least two members showed significant stoichiometry change (adj. p < 0.05 and absolute log2 fold change > 0.5). The complete list of stoichiometry changes is available in Dataset EV6.Violin plots depicting interquartile ranges (IQRs) of individual members of protein complexes during aging. The IQR for each protein complex considered in G was calculated using transcript (total RNA dataset, light blue) or protein (dark blue) log2 fold changes between two age groups (n=4 per age group for proteome and n=5 per age group for transcriptome). * p < 0.05, Wilcoxon Rank Sum test. The central line of the violin plots indicates the median value.Heat map showing relative protein fold changes for members of selected complexes affected by aging. Names of significantly affected members in the 39 wph vs. 12 wph comparison (adj. p < 0.05 and absolute log2 fold change > 0.5) are highlighted in bold with a star. Related to Figure EV3 and Dataset EV5 and EV6.