Figure 6 ( A ) Representative photopic ERG flicker series at 1 , 5 and 10 Hz with 3 cd . s / m2 illumination at 10 WPI . Arrowheads indicate light stimulus . ( B ) Representative photopic single flash ERG signal with 1 , 3 and 10 cd . s / m2 illumination . The vertical dotted line indicates light stimulus . ( C ) Quantification of photopic B - wave amplitude from single flash responses to 1 , 3 and 10 cd . s / m2 illumination of wildtype C57BL6 / J x 129 / sv eyes ( WT , n = 3 ) compared to treated eyes ( IVT / GL , n ≥ 3 ) and untreated eyes ( UN , n ≥ 3 ) at 10 weeks post injection . ( E - F ) Confocal images of sections proximal to the injection site in Q2 ( E ) and distal to the injection site in Q4 ( F ) , showing Cnga3 expression in PNA - positive cone outer segments . OS , outer segment layer ; ONL , outer nuclear layer ; INL , inner nuclear layer . ( G - I ) Confocal images of Cnga3 - / - treated ( G ) , wildtype ( H ) and Cnga3 - / - untreated ( I ) retinal sections stained for Cnga3 . Note some non - specific signal detected in INL and adjacent synaptic layer . Scale bar : 50 μm . OS , outer segment layer ; ONL , outer nuclear layer ; INL , inner nuclear layer .

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

Figure 6(A) Representative photopic ERG flicker series at 1, 5 and 10 Hz with 3 cd.s/m2 illumination at 10 WPI. Arrowheads indicate light stimulus.(B) Representative photopic single flash ERG signal with 1, 3 and 10 cd.s/m2 illumination. The vertical dotted line indicates light stimulus. (C) Quantification of photopic B-wave amplitude from single flash responses to 1, 3 and 10 cd.s/m2 illumination of wildtype C57BL6/J x 129/sv eyes (WT, n = 3) compared to treated eyes (IVT/GL, n ≥ 3) and untreated eyes (UN, n ≥ 3) at 10 weeks post injection. (E-F) Confocal images of sections proximal to the injection site in Q2 (E) and distal to the injection site in Q4 (F), showing Cnga3 expression in PNA-positive cone outer segments. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.(G-I) Confocal images of Cnga3-/- treated (G), wildtype (H) and Cnga3-/- untreated (I) retinal sections stained for Cnga3. Note some non-specific signal detected in INL and adjacent synaptic layer. Scale bar: 50 μm. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.


Figure 1C - D ) Representative western blots and respective densitometries ( right panel ) for the selected SWI / SNF / BAF complex subunits ( n = 3 - 5 biological replicates ) . Arrow heads indicate non - specific bands , while # symbol highlights BAF53B . F ) Representative immunoblots of co - immunoprecipitation ( co - IP ) experiments in eNPCs ( top panels ) and smNPCs ( bottom panels ) . Co - IPs were performed with antibodies against BAF170 ( left ) and BRG1 ( right ) . Inputs are homogenates from eNPCs ( left ) and smNPCs ( right ) . IgG indicates IgG - bound Sepharose beads ( as controls ) , while IP indicates immunoprecipitated samples after elution from the beads . Arrow head indicates BCL7A band at the correct MW . G ) Immunofluorescence staining of BCL7A ( purple ) along with actin , nestin and β - III tubulin ( green ) as well as GFAP ( white ) in iPSCs , smNPCs , and smNPC - derived neurons and astrocytes , respectively . Arrow heads indicate GFAP - positive glial cells with low BCL7A expression . H ) Western blot analysis of BCL7A and BAF170 in cortex and hippocampus of embryonic ( E17 . 5 ) , early postnatal ( PD0 and PD7 ) and adult ( PD120 ) mouse brain tissue showing a progressive temporal reduction of protein levels in the analyzed tissues . I ) Immunofluorescence staining of BCL7A ( purple ) , EdU ( green ) and NeuN ( white ) in embryonic ( E17 . 5 ) and adult ( PD120 ) hippocampal brain sections . EdU was used to label proliferating NPCs , whereas NeuN was used as a neuronal marker .

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

Figure 1C-D) Representative western blots and respective densitometries (right panel) for the selected SWI/SNF/BAF complex subunits (n=3-5 biological replicates). Arrow heads indicate non-specific bands, while # symbol highlights BAF53B.F) Representative immunoblots of co-immunoprecipitation (co-IP) experiments in eNPCs (top panels) and smNPCs (bottom panels). Co-IPs were performed with antibodies against BAF170 (left) and BRG1(right). Inputs are homogenates from eNPCs (left) and smNPCs (right). IgG indicates IgG-bound Sepharose beads (as controls), while IP indicates immunoprecipitated samples after elution from the beads. Arrow head indicates BCL7A band at the correct MW.G) Immunofluorescence staining of BCL7A (purple) along with actin, nestin and β-III tubulin (green) as well as GFAP (white) in iPSCs, smNPCs, and smNPC-derived neurons and astrocytes, respectively. Arrow heads indicate GFAP-positive glial cells with low BCL7A expression.H) Western blot analysis of BCL7A and BAF170 in cortex and hippocampus of embryonic (E17.5), early postnatal (PD0 and PD7) and adult (PD120) mouse brain tissue showing a progressive temporal reduction of protein levels in the analyzed tissues.I) Immunofluorescence staining of BCL7A (purple), EdU (green) and NeuN (white) in embryonic (E17.5) and adult (PD120) hippocampal brain sections. EdU was used to label proliferating NPCs, whereas NeuN was used as a neuronal marker.


Figure 2A ) Immunoblot and immunofluorescence staining of BCL7A ( red ) in eNPCs isolated from Bcl7awt / wt ( wt ) and Bcl7ako / ko ( ko ) embryos ( E13 . 5 ) . Nestin ( green ) was used as NPC marker , whereas DAPI ( blue ) was used to stain nuclei . B ) Immunoblot and immunofluorescence staining of BCL7A in smNPCs . Two different BCL7A knockout clones ( KO1 and KO2 ) were used along with the parental ( P ) cell line . C - D ) Representative western blots and respective densitometries ( right panel ) for selected SWI / SNF / BAF complex subunits in ( C ) eNPCs ( n = 4 biological replicates ) and ( D ) smNPCs ( n = 3 - 6 biological replicates ) . E - G ) BCL7A KO and wt smNPCs were used for ( F ) nuclear isolation followed by density sedimentation Top panels show representative immunoblots for selected SWI / SNF / BAF complex subunits and respective densitometries are shown below ( n = 3 biological replicates ) . BCL7A KO and wt smNPCs were used for differential salt extraction . Top panels show representative immunoblots for selected SWI / SNF / BAF complex subunits and respective densitometries are shown below ( n = 3 biological replicates ) . H - I ) Heatmaps of SMARCA4 / BRG1 and H3K27me3 ChIP - seq in wt and BCL7A KO eNPCs . J ) Average density plots for SMARCA4 / BRG1 occupancy around the TSS in wt and BCL7A KO eNPCs . K - L ) Heatmaps of SMARCA4 / BRG1 and H3K27me3 ChIP - seq in parental and BCL7A KO smNPCs . M ) Average density plots for SMARCA4 / BRG1 occupancy around the TSS in parental and BCL7A KO smNPCs .

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

Figure 2A) Immunoblot and immunofluorescence staining of BCL7A (red) in eNPCs isolated from Bcl7awt/wt (wt) and Bcl7ako/ko (ko) embryos (E13.5). Nestin (green) was used as NPC marker, whereas DAPI (blue) was used to stain nuclei.B) Immunoblot and immunofluorescence staining of BCL7A in smNPCs. Two different BCL7A knockout clones (KO1 and KO2) were used along with the parental (P) cell line.C-D) Representative western blots and respective densitometries (right panel) for selected SWI/SNF/BAF complex subunits in (C) eNPCs (n=4 biological replicates) and (D) smNPCs (n=3-6 biological replicates).E-G) BCL7A KO and wt smNPCs were used for (F) nuclear isolation followed by density sedimentation Top panels show representative immunoblots for selected SWI/SNF/BAF complex subunits and respective densitometries are shown below (n=3 biological replicates).BCL7A KO and wt smNPCs were used for differential salt extraction. Top panels show representative immunoblots for selected SWI/SNF/BAF complex subunits and respective densitometries are shown below (n=3 biological replicates).H-I) Heatmaps of SMARCA4/BRG1 and H3K27me3 ChIP-seq in wt and BCL7A KO eNPCs. J) Average density plots for SMARCA4/BRG1 occupancy around the TSS in wt and BCL7A KO eNPCs.K-L) Heatmaps of SMARCA4/BRG1 and H3K27me3 ChIP-seq in parental and BCL7A KO smNPCs. M) Average density plots for SMARCA4/BRG1 occupancy around the TSS in parental and BCL7A KO smNPCs.


Figure 3A ) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs . Samples were collected at the indicated differentiation time points . Respective densitometries are shown on the right ( n = 6 - 7 biological replicates ) . B ) Sholl analysis of β - III tubulin - positive immature wt and BCL7A KO neurons following 7 days of spontaneous eNPC differentiation ( n = 4 biological replicates , each experiment with 10 - 15 cells per condition ) . C ) Immunofluorescence staining ( central panel ) and quantification ( right panel ) of β - III tubulin - and GFAP - positive cells as well as RT - PCR analysis ( right panels ) of spontaneously differentiated iPSC - derived parental and BCL7A KO smNPCs ( n = 3 - 7 biological replicates ) . D ) Immunofluorescence staining of BCL7A expression in hippocampal sections from adult control and Bcl7afl / fl ; Nestin - Cretg / wt mice . E ) EdU experiments in adult mice . Animals were injected with EdU twice daily for three days and sacrificed 21 days thereafter . Images show immunofluorescence stainings for EdU ( green ) , doublecortin ( DCX , white ) , and S100β ( red ) in hippocampal brain sections from Bcl7awt / wt ; Nestin - Cretgt / wt ( as control ) and Bcl7afl / fl ; Nestin - Cretg / wt mice . Representative EdU + DCX + ( yellow triangles ) or EdU + S100β + ( yellow asterisk ) double - labelled cells are indicated in the insets . Scale bar = 50 μm and 20 μm ( for insets ) . F ) Quantification of EdU + ( right panel ) , EdU + DCX + ( middle panel ) and EdU + S100β + ( left panel ) cells within the hippocampal dentate gyrus ( DG ) of adult control ( n = 4 ) and Bcl7afl / fl ; Nestin - Cretg / wt ( n = 5 ) animals . G ) Immunofluorescence staining for DCX + cells within the DG region of adult Bcl7afl / fl ; Nestin - Crewt / wt ( as control ) and Bcl7afl / fl ; Nestin - Cretg / wt animals . Confocal imaging analysis shows a lower number , misalignment and reduced neuritic complexity of DCX + labelled cells in BCL7A KO mice compared to controls . Yellow arrows indicate misaligned DCX + cells . H ) Immunofluorescence staining of BCL7A ( red ) of hippocampal sections from Bcl7afl / fl ; Baf53b - Crewt / wt ( as control ) and Bcl7afl / fl ; Baf53b - Cretg / wt mice . I ) Immunofluorescence staining of BCL7A ( red ) and DCX ( green ) in hippocampal sections of control and Bcl7afl / fl ; Baf53b - Cretg / wt mice . Yellow arrow indicates dividing cells , whereas yellow asterisks mark differentiating immature neurons . J ) Immunofluorescence staining for DCX ( white ) within the DG region of adult control and Bcl7afl / fl ; Baf53b - Cretg / wt mice . Representative images show no obvious difference in number , morphology or alignment of DCX + labelled cells . Quantification of DCX + labelled cells in control and Bcl7afl / fl ; Baf53b - Cretg / wt mice is shown on the right ( n = 3 per genotype ) .

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

Figure 3A) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs. Samples were collected at the indicated differentiation time points. Respective densitometries are shown on the right (n=6-7 biological replicates).B) Sholl analysis of β-III tubulin-positive immature wt and BCL7A KO neurons following 7 days of spontaneous eNPC differentiation (n=4 biological replicates, each experiment with 10-15 cells per condition).C) Immunofluorescence staining (central panel) and quantification (right panel) of β-III tubulin- and GFAP-positive cells as well as RT-PCR analysis (right panels) of spontaneously differentiated iPSC-derived parental and BCL7A KO smNPCs (n=3-7 biological replicates).D) Immunofluorescence staining of BCL7A expression in hippocampal sections from adult control and Bcl7afl/fl; Nestin-Cretg/wt mice.E) EdU experiments in adult mice. Animals were injected with EdU twice daily for three days and sacrificed 21 days thereafter. Images show immunofluorescence stainings for EdU (green), doublecortin (DCX, white), and S100β (red) in hippocampal brain sections from Bcl7awt/wt; Nestin-Cretgt/wt (as control) and Bcl7afl/fl; Nestin-Cretg/wt mice. Representative EdU+ DCX+ (yellow triangles) or EdU+ S100β+ (yellow asterisk) double-labelled cells are indicated in the insets. Scale bar= 50 μm and 20 μm (for insets).F) Quantification of EdU+ (right panel), EdU+ DCX+ (middle panel) and EdU+ S100β+ (left panel) cells within the hippocampal dentate gyrus (DG) of adult control (n=4) and Bcl7afl/fl; Nestin-Cretg/wt (n=5) animals.G) Immunofluorescence staining for DCX+ cells within the DG region of adult Bcl7afl/fl; Nestin-Crewt/wt (as control) and Bcl7afl/fl; Nestin-Cretg/wt animals. Confocal imaging analysis shows a lower number, misalignment and reduced neuritic complexity of DCX+ labelled cells in BCL7A KO mice compared to controls. Yellow arrows indicate misaligned DCX+ cells.H) Immunofluorescence staining of BCL7A (red) of hippocampal sections from Bcl7afl/fl; Baf53b-Crewt/wt (as control) and Bcl7afl/fl; Baf53b-Cretg/wt mice.I) Immunofluorescence staining of BCL7A (red) and DCX (green) in hippocampal sections of control and Bcl7afl/fl; Baf53b-Cretg/wt mice. Yellow arrow indicates dividing cells, whereas yellow asterisks mark differentiating immature neurons.J) Immunofluorescence staining for DCX (white) within the DG region of adult control and Bcl7afl/fl; Baf53b-Cretg/wt mice. Representative images show no obvious difference in number, morphology or alignment of DCX+ labelled cells. Quantification of DCX+ labelled cells in control and Bcl7afl/fl; Baf53b-Cretg/wt mice is shown on the right (n=3 per genotype).


Figure 4B - C ) Representative Ca2 + measurements in ( B ) wt vs BCL7A KO cortical neurons ( CN , 10 days in vitro ) and in ( C ) smNPC - derived parental and BCL7A KO neurons . Ca2 + changes were detected using Fluo - 4 and upon treatment with ( B ) 50 μM glutamate ( final concentration ) and ( C ) 100 μM glutamate ( final concentration ) . Upper right panels report Ca2 + peaks ( F ) relative to baseline ( F0 ) . In B , wt : n = 18 cells ; ko : n = 23 cells ( n = 2 independent experiments ) . In C , P : n = 27 cells ; KO1 : n = 20 cells ; KO2 : n = 19 cells ( n = 2 independent experiments ) . D ) Immunoblot analysis of hippocampal tissues . Densitometries are shown on the right ( ctr : n = 4 - 5 mice ; ko : n = 3 - 4 mice ) . E ) RT - PCR analysis of FOS expression upon glutamate ( 100 μM for 10 min ) stimulation in smNPC - derived parental and BCL7A KO neurons ( n = 5 - 6 biological replicates ) . F ) Immunofluorescence staining of cFos in the dentate gyrus of adult Bcl7afl / fl ; Baf53b - Cretg / wt ( ko ) mice and control ( ctr ) littermates . Mice were either kept in standard housing conditions ( upper images ; ctr : n = 3 mice ; ko : n = 3 mice ) or exposed to an enriched environment ( lower images ; ctr : n = 3 mice ; ko : n = 4 mice ) . Quantification of cFos + cells are shown below . G - K ) Behavioral characterization of Bcl7afl / fl ; Nestin - Cretg / wt ( ko ) mice compared to control ( ctr ) littermates . Animals were tested for their locomotor activity and coordination in ( H ) the open field ( ctr : n = 13 mice ; ko : n = 11 mice ) and ( I ) RotaRod test ( ctr : n = 12 mice ; ko : n = 7 mice ) . Cognitive function was assessed in ( J ) the Y - maze spontaneous alternation task ( ctr : n = 13 mice ; ko : n = 11 mice ) and ( K ) fear conditioning paradigm ( ctr : n = 11 mice ; ko : n = 10 mice ) . The percentage of spontaneous alternations and freezing were used as measures for working and long - term memory function , respectively . L - Q ) Behavioral characterization of adult Bcl7afl / fl ; Baf53b - Cretg / wt ( nKO ) mice compared to control ( nKO ) littermates . Locomotor activity and coordination were assessed in ( M ) the open field ( ctr : n = 14 mice ; nKO : n = 12 mice ) and ( N ) RotaRod paradigm ( ctr : n = 16 mice ; nKO : n = 12 mice ) . Cognitive function was tested in ( O ) the Y - maze ( ctr : n = 11 mice ; nKO : n = 11 mice ) and ( P - Q ) Barnes maze test ( ctr : n = 11 mice ; nKO : n = 11 mice ) . The percentage of spontaneous alternations and freezing were used as measures for working and long - term memory function , respectively . Representative heatmaps of Barnes maze test are reported on the right . The time spent in target ( T ) and non - target quadrants during the probe trial are shown on the right .

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 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 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 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 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

Figure 4B-C) Representative Ca2+ measurements in (B) wt vs BCL7A KO cortical neurons (CN, 10 days in vitro) and in (C) smNPC-derived parental and BCL7A KO neurons. Ca2+ changes were detected using Fluo-4 and upon treatment with (B) 50 μM glutamate (final concentration) and (C) 100 μM glutamate (final concentration). Upper right panels report Ca2+ peaks (F) relative to baseline (F0). In B, wt: n=18 cells; ko: n=23 cells (n=2 independent experiments). In C, P: n=27 cells; KO1: n=20 cells; KO2: n=19 cells (n=2 independent experiments).D) Immunoblot analysis of hippocampal tissues. Densitometries are shown on the right (ctr: n=4-5 mice; ko: n=3-4 mice).E) RT-PCR analysis of FOS expression upon glutamate (100 μM for 10 min) stimulation in smNPC-derived parental and BCL7A KO neurons (n=5-6 biological replicates).F) Immunofluorescence staining of cFos in the dentate gyrus of adult Bcl7afl/fl; Baf53b-Cretg/wt (ko) mice and control (ctr) littermates. Mice were either kept in standard housing conditions (upper images; ctr: n=3 mice; ko: n=3 mice) or exposed to an enriched environment (lower images; ctr: n=3 mice; ko: n=4 mice). Quantification of cFos+ cells are shown below.G-K) Behavioral characterization of Bcl7afl/fl; Nestin-Cretg/wt (ko) mice compared to control (ctr) littermates. Animals were tested for their locomotor activity and coordination in (H) the open field (ctr: n=13 mice; ko: n=11 mice) and (I) RotaRod test (ctr: n=12 mice; ko: n=7 mice). Cognitive function was assessed in (J) the Y-maze spontaneous alternation task (ctr: n=13 mice; ko: n=11 mice) and (K) fear conditioning paradigm (ctr: n=11 mice; ko: n=10 mice). The percentage of spontaneous alternations and freezing were used as measures for working and long-term memory function, respectively.L-Q) Behavioral characterization of adult Bcl7afl/fl; Baf53b-Cretg/wt (nKO) mice compared to control (nKO) littermates. Locomotor activity and coordination were assessed in (M) the open field (ctr: n=14 mice; nKO: n=12 mice) and (N) RotaRod paradigm (ctr: n=16 mice; nKO: n=12 mice). Cognitive function was tested in (O) the Y-maze (ctr: n=11 mice; nKO: n=11 mice) and (P-Q) Barnes maze test (ctr: n=11 mice; nKO: n=11 mice). The percentage of spontaneous alternations and freezing were used as measures for working and long-term memory function, respectively. Representative heatmaps of Barnes maze test are reported on the right. The time spent in target (T) and non-target quadrants during the probe trial are shown on the right.


Figure 5A ) Heatmap of significantly dysregulated genes in BCL7A KO compared to wt eNPCs . E ) Heatmap of significantly dysregulated genes encoding components of the Wnt and Notch signaling pathways . F ) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs in presence of 1 μM DAPT or 3 μM CHIR99021 . G ) Immunofluorescence staining of eNPCs undergoing spontaneous differentiation for 7 days . BCL7A KO eNPCs were incubated for 72 h ( 24 h in proliferation plus 48 h in differentiation medium ) with DMSO , 1 μM DAPT , 3 μM CHIR99021 or a combination of 1 μM DAPT + 3 μM CHIR99021 . Quantification of the β - III tubulin - positive or GFAP - positive cells is shown on the right ( n = 3 - 6 technical replicates from 3 independent experiments ) . H ) Sholl analysis of β - III tubulin - positive immature neurons after 7 days of spontaneous differentiation ( n = 4 biological replicates with > 30 cells per condition ) . I ) mRNA expression levels of TUBB3 and GFAP at 15 and 25 days of spontaneously differentiating BCL7A KO compared parental smNPCs . BCL7A KO smNPCs were treated with DAPT , CHIR99021 , DAPT + CHIR99021 or DMSO ( as control = ctr ) during the initial 7 days of spontaneous differentiation ( n = 3 - 7 biological replicates ) . J ) Immunoblots for non - phospho ( active ) β - catenin and TCF4 / TCF7L2 in proliferating and 48 h - differentiated wt and BCL7A KO eNPCs . Respective densitometries are shown on the right ( n = 5 - 8 biological replicates per condition ) .

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Figure 5A) Heatmap of significantly dysregulated genes in BCL7A KO compared to wt eNPCs.E) Heatmap of significantly dysregulated genes encoding components of the Wnt and Notch signaling pathways.F) Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs in presence of 1 μM DAPT or 3 μM CHIR99021.G) Immunofluorescence staining of eNPCs undergoing spontaneous differentiation for 7 days. BCL7A KO eNPCs were incubated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with DMSO, 1 μM DAPT, 3 μM CHIR99021 or a combination of 1 μM DAPT + 3 μM CHIR99021. Quantification of the β-III tubulin-positive or GFAP-positive cells is shown on the right (n=3-6 technical replicates from 3 independent experiments).H) Sholl analysis of β-III tubulin-positive immature neurons after 7 days of spontaneous differentiation (n=4 biological replicates with >30 cells per condition).I) mRNA expression levels of TUBB3 and GFAP at 15 and 25 days of spontaneously differentiating BCL7A KO compared parental smNPCs. BCL7A KO smNPCs were treated with DAPT, CHIR99021, DAPT + CHIR99021 or DMSO (as control= ctr) during the initial 7 days of spontaneous differentiation (n=3-7 biological replicates).J) Immunoblots for non-phospho (active) β-catenin and TCF4/TCF7L2 in proliferating and 48 h-differentiated wt and BCL7A KO eNPCs. Respective densitometries are shown on the right (n=5-8 biological replicates per condition).


Figure 6G ) Proximity ligation assay ( PLA ) for the OXPHOS subunits NDUFB8 and MTCO1 in adult hippocampal sections from Bcl7afl / fl ; Baf53b - Cretg / wt ( ko ; n = 4 ) and control littermates ( ctr ; n = 4 ) . Sections were co - stained with TOM20 and NeuN to label mitochondria and neurons , respectively . The total number of PLA dots was normalized to the mitochondrial area ( panel on the lower right ) .

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Figure 6G) Proximity ligation assay (PLA) for the OXPHOS subunits NDUFB8 and MTCO1 in adult hippocampal sections from Bcl7afl/fl; Baf53b-Cretg/wt (ko; n=4) and control littermates (ctr; n=4). Sections were co-stained with TOM20 and NeuN to label mitochondria and neurons, respectively. The total number of PLA dots was normalized to the mitochondrial area (panel on the lower right).


Figure 7A ) OCR measurements in wt and BCL7A KO eNPCs treated with 3 μM of CHIR99021 or DMSO ( as control , ctr ) for 24 h . The average maximal respiration upon FCCP treatment is shown on the right ( n = 5 - 7 biological replicates ) . B ) OCR measurements in wt and BCL7A KO eNPCs following 48h of spontaneous differentiation . BCL7A KO eNPCs were treated for 72 h ( 24 h in proliferation plus 48 h in differentiation medium ) with CHIR99021 or DMSO . The average maximal respiration upon FCCP treatment is shown on the right ( n = 4 biological replicates ) . C ) Immunoblot analysis of differentiated BCL7A KO eNPCs exposed to 3 μM CHIR99021 , in presence or absence of 20 nM rotenone ( rot ) . Antibodies against β - III tubulin and actin were used . D ) Immunofluorescence staining of spontaneously differentiated eNPCs . BCL7A KO eNPCs were treated for 72 h ( i . e . , 24 h in proliferation plus 48 h in differentiation medium ) with 3 μM CHIR99021 , in presence or absence of 20 nM rotenone . Scale bar = 50 μm . Statistics are shown on the right ( n = 3 - 6 technical replicates from 3 independent experiments ) . E ) Sholl analysis of β - III tubulin - positive immature neurons following 7 days of spontaneous differentiation . BCL7A KO eNPCs were exposed to 3 μM CHIR99021 , in presence or absence of 20 nM rotenone ( n = 3 biological replicates ) . F ) Representative OCR measurements in BCL7A KO eNPCs treated with 5 and 10 μM of pioglitazone or DMSO ( as control , ctr ) for 24 h . The average maximal respiration upon FCCP treatment is shown on the right ( n = 3 biological replicates ) . G ) Immunofluorescence staining of spontaneously differentiated eNPCs . BCL7A KO eNPCs were treated for 72 h ( i . e . , 24 h in proliferation plus 48 h in differentiation medium ) with 5 μM pioglitazone . The percentage of β - III - tubulin - positive cells is shown on the right ( n = 3 - 8 technical replicates from 2 independent experiments ) .

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

Figure 7A) OCR measurements in wt and BCL7A KO eNPCs treated with 3 μM of CHIR99021 or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 5-7 biological replicates).B) OCR measurements in wt and BCL7A KO eNPCs following 48h of spontaneous differentiation. BCL7A KO eNPCs were treated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with CHIR99021 or DMSO. The average maximal respiration upon FCCP treatment is shown on the right (n= 4 biological replicates).C) Immunoblot analysis of differentiated BCL7A KO eNPCs exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (rot). Antibodies against β-III tubulin and actin were used.D) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 3 μM CHIR99021, in presence or absence of 20 nM rotenone. Scale bar= 50 μm. Statistics are shown on the right (n=3-6 technical replicates from 3 independent experiments).E) Sholl analysis of β-III tubulin-positive immature neurons following 7 days of spontaneous differentiation. BCL7A KO eNPCs were exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (n=3 biological replicates).F) Representative OCR measurements in BCL7A KO eNPCs treated with 5 and 10 μM of pioglitazone or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 3 biological replicates).G) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 5 μM pioglitazone. The percentage of β-III-tubulin-positive cells is shown on the right (n=3-8 technical replicates from 2 independent experiments).


Figure 2C , D , E Examples of embryos expressing the tFT - Bcd reporter with C : mCherry - sfGFP - Bcd , D : fmCherry - sfGFP - Bcd , and E : mCherry - sfGFP . For all , ( i ) Images of " shells " of embryos in early n . c . 14 . 3D images of the embryos were generated , and then the interior of the embryo was erased , leaving only the embryo cortex to improve clarity ( ii ) Mean AP intensity profile of each color for the embryo in i . Shade region represents ± 1 s . d . Inset shows same profiles after multiplication of red intensity by constant factor . Data binned into 10 µm bins ( n = 4 embryos ) . ( iii ) Ratio of green over red signal , reflecting protein age . The thin lines represent individual embryos , while the thick solid line is the mean . The solid dashed line depicts the mean green / red ratio for a line with the tFT - Bcd reporter but lacking endogenous Bcd ( BcdE1 mutant , n = 4 ) .

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Figure 2C,D,E Examples of embryos expressing the tFT-Bcd reporter with C: mCherry-sfGFP-Bcd, D: fmCherry-sfGFP-Bcd, and E: mCherry-sfGFP. For all, (i) Images of "shells" of embryos in early n.c. 14. 3D images of the embryos were generated, and then the interior of the embryo was erased, leaving only the embryo cortex to improve clarity (ii) Mean AP intensity profile of each color for the embryo in i. Shade region represents ±1 s.d. Inset shows same profiles after multiplication of red intensity by constant factor. Data binned into 10 µm bins (n=4 embryos). (iii) Ratio of green over red signal, reflecting protein age. The thin lines represent individual embryos, while the thick solid line is the mean. The solid dashed line depicts the mean green/red ratio for a line with the tFT-Bcd reporter but lacking endogenous Bcd (BcdE1 mutant, n=4).


Figure 4 , B , C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd ( gray , open squares , N = 10 ) . Control embryos were injected with DMSO ( black , filled circles , N = 9 ) . All embryos are y / w and were injected while in cycle 14 and imaged 60 min later . D The age of Bcd and its gradient are unaltered in fsd mutant . Comparison of a representative y / w embryo ( left ) and loss - of - function mutation fsdKG02393 ( right ) are shown . Both embryos have the tFT - Bcd construct in the same locus , are in early n . c . 14 , were imaged side - by - side under identical conditions , and are displayed with the same settings . Embryo " shells " are displayed . E Quantification of embryos Fluorescence intensity from sfGFP ( green ) and mCherry ( magenta ) for wt ( N = 8 , filled circles ) and fsd ( N = 6 , open squares ) at early cycle 14 . Inset : tFT - Bcd ratio for wt ( black ) and FSD ( gray ) embryos .

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Figure 4,B,C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd (gray, open squares, N=10). Control embryos were injected with DMSO (black, filled circles, N=9). All embryos are y/w and were injected while in cycle 14 and imaged 60 min later.D The age of Bcd and its gradient are unaltered in fsd mutant. Comparison of a representative y/w embryo (left) and loss-of-function mutation fsdKG02393 (right) are shown. Both embryos have the tFT-Bcd construct in the same locus, are in early n.c. 14, were imaged side-by-side under identical conditions, and are displayed with the same settings. Embryo "shells" are displayed.E Quantification of embryos Fluorescence intensity from sfGFP (green) and mCherry (magenta) for wt (N=8, filled circles) and fsd (N=6, open squares) at early cycle 14. Inset: tFT-Bcd ratio for wt (black) and FSD (gray) embryos.


Figure 5Time - course of a representative y / w embryo with the tFT - Bcd reporter from the division cycle 10 to the early gastrulation . A SPIM images of the embryo early development . Embryo " shells " are displayed . Left : Green fluorescence , center : Red fluorescence , right : Intensity - weighted ratiometric image of the mCherry / sfGFP ratio , reporting average protein age . The scale corresponds to 50 μm . B Quantification of the same embryo as is A . Each line represents the fluorescence profile along the anterior - posterior axis of the embryo for a time frame ( separated by 6 min ) . In the lower panel , where the mCherry / sfGFP ratio is shown , the posterior half of the embryo is not shown because the signal drops to the background levels ( due to softer imaging conditions for the long term time - course ) . C The same data is shown as a function of time for different embryo segments .

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Figure 5Time-course of a representative y/w embryo with the tFT-Bcd reporter from the division cycle 10 to the early gastrulation. A SPIM images of the embryo early development. Embryo "shells" are displayed. Left: Green fluorescence, center: Red fluorescence, right: Intensity-weighted ratiometric image of the mCherry/sfGFP ratio, reporting average protein age. The scale corresponds to 50 μm. B Quantification of the same embryo as is A. Each line represents the fluorescence profile along the anterior-posterior axis of the embryo for a time frame (separated by 6 min). In the lower panel, where the mCherry/sfGFP ratio is shown, the posterior half of the embryo is not shown because the signal drops to the background levels (due to softer imaging conditions for the long term time-course). C The same data is shown as a function of time for different embryo segments.


Figure 1C . Aliquots of the supernatants of the indicated HEK293T transfectants ( all expressing λ chains ) were resolved under non - reducing conditions and blotted with anti - µ antibodies . Wild type µs chains are secreted only as pentamers and hexamers , whilst mutants lacking C575 release mostly µs2L2 subunits ( indicated with a red arrow ) as does the double C414A - C575A . L566A releases instead mainly µs2L2 subunits of faster migration ( blue arrow ) . C414 mutants release no hexamers , but smaller assemblies , including fast migrating µs2L2 subunits . E . Aliquots of the supernatants were treated with or without Endo - H and electrophoresed under reducing conditions . The blot was decorated with anti - µ antibodies . N563 - glycans acquire Endo - H resistance in µsL566A and µsC575A , but remain largely sensitive in wild type µs .

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Figure 1C. Aliquots of the supernatants of the indicated HEK293T transfectants (all expressing λ chains) were resolved under non-reducing conditions and blotted with anti-µ antibodies. Wild type µs chains are secreted only as pentamers and hexamers, whilst mutants lacking C575 release mostly µs2L2 subunits (indicated with a red arrow) as does the double C414A-C575A. L566A releases instead mainly µs2L2 subunits of faster migration (blue arrow). C414 mutants release no hexamers, but smaller assemblies, including fast migrating µs2L2 subunits.E. Aliquots of the supernatants were treated with or without Endo-H and electrophoresed under reducing conditions. The blot was decorated with anti-µ antibodies. N563-glycans acquire Endo-H resistance in µsL566A and µsC575A, but remain largely sensitive in wild type µs.


Figure 2A . Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non - reducing conditions before or after removal of N - glycans by PNGaseF . Blots were decorated with anti - µ antibodies . Blue and red arrows point to the closed ( oxidized ) and open µs2L2 species respectively . Intracellular µs2L2 species purified from the murine hybridoma B1 . 8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS / MS spectrum Y and B ions are shown in blue and red , respectively .

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Figure 2A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively.Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS/MS spectrum Y and B ions are shown in blue and red, respectively.


Figure 3A . I . 29µ + B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion . At the indicated times , cells were lysed in TritonX114 to separate soluble and integral membrane proteins ( aqueous and detergent phases , respectively ) . Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti - µ antibodies . The blue arrow points at a band likely corresponding to µs2L2C575OX , the intensity of which increases upon LPS stimulation . C . µ - chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM . D . The bar graphs show the levels of C575 and C414 oxidation , expressed as the ratio between IAA and NEM modified peptides .

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Figure 3A. I.29µ+ B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion. At the indicated times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to µs2L2C575OX, the intensity of which increases upon LPS stimulation.C. µ-chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM.D. The bar graphs show the levels of C575 and C414 oxidation, expressed as the ratio between IAA and NEM modified peptides.


Figure 4ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . A . Aliquots from the spent media ( Extracellular ) were blotted under non - reducing conditions and decorated with anti - µ antibodies . The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575 - dependent retention , and wild type µs are secreted only as polymers ( black arrow ) . Traces of covalent HaloERp44 - µ2L2 complexes ( black asterisks ) are detected in the media of cells expressing HaloERp44 ∆ RDEL and µs or L566A . The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44 ∆ RDEL suggest that the latter inhibits processing of the N563 glycan , likely hindering it to the Golgi enzymes upon non - covalent binding . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition . ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . B . The corresponding cell lysates are shown in this panel . The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44 . The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2 . Traces of intracellular µs2L2 - ERp44 ∆ RDEL complexes are visible in µs and L566A but absent from C575A expressing cells . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition . C . Lysates of the ERp44KO transfectants were precipitated with NP - sepharose , resolved under non reducing conditions and the blot decorated sequentially with anti - µ , anti - ERp44 or anti - Halo . The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo - tagged ERp44 .

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Figure 4ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. B. The corresponding cell lysates are shown in this panel. The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44. The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2. Traces of intracellular µs2L2-ERp44∆RDEL complexes are visible in µs and L566A but absent from C575A expressing cells. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.C. Lysates of the ERp44KO transfectants were precipitated with NP-sepharose, resolved under non reducing conditions and the blot decorated sequentially with anti-µ, anti-ERp44 or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.


Figure 5B . To selectively label intracellular HC575A , cells were treated with 1 nM Halo - tag TMR Direct Ligand for 24 hours . The left panel shows aliquots of the lysates of the indicated transfectants , resolved under non - reducing conditions and decorated with anti - λ antibodies . In the two right panels , samples were precipitated with NP - Sepharose and electrophoresed under non reducing conditions . The signal for the Halo - tag TMR was checked directly in the gel . The arrows point at the various assemblies between µ , HC575A and λ chains . C . The experiment shown aims to determine whether ERp44 can bind also to a single C575 , or rather to non - native bonds . Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP - Sepharose , resolved under non reducing conditions and the blot decorated with anti - λ antibodies or anti - ERp44 as indicated . Black asterisks point to covalent complexes of Halo - ERp44 with µs2L2 .

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Figure 5B. To selectively label intracellular HC575A, cells were treated with 1 nM Halo-tag TMR Direct Ligand for 24 hours. The left panel shows aliquots of the lysates of the indicated transfectants, resolved under non-reducing conditions and decorated with anti-λ antibodies. In the two right panels, samples were precipitated with NP-Sepharose and electrophoresed under non reducing conditions. The signal for the Halo-tag TMR was checked directly in the gel. The arrows point at the various assemblies between µ, HC575A and λ chains.C. The experiment shown aims to determine whether ERp44 can bind also to a single C575, or rather to non-native bonds. Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP-Sepharose, resolved under non reducing conditions and the blot decorated with anti-λ antibodies or anti-ERp44 as indicated. Black asterisks point to covalent complexes of Halo-ERp44 with µs2L2.


Figure 1 ( A ) Quantification of CXCR4 + cells by flow cytometry and Sox17 + cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH . Average and standard deviation ( SD ) of eight independent experiments . ( B ) Gene expression profiling by RT - PCR at day 7 differentiation . Average and SD of three independent experiments . ( C ) Immunostaining for Ecadherin ( left ) and FoxA2 ( right ) at day 7 of differentiation in presence of 3 µM CH . ( D ) Quantification of CXCR4 + by flow cytometry over a seven - day differentiation time course in the presence of 3 µM CH . Average and SD of three independent experiments . ( E ) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH . Scale bars = 200

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Figure 1(A) Quantification of CXCR4+ cells by flow cytometry and Sox17+ cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH. Average and standard deviation (SD) of eight independent experiments.(B) Gene expression profiling by RT-PCR at day 7 differentiation. Average and SD of three independent experiments.(C) Immunostaining for Ecadherin (left) and FoxA2 (right) at day 7 of differentiation in presence of 3 µM CH.(D) Quantification of CXCR4+ by flow cytometry over a seven-day differentiation time course in the presence of 3 µM CH. Average and SD of three independent experiments.(E) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH. Scale bars = 200 µM


Figure 2 ( A ) Assay of Tcf7l1 mRNA by RT - PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH . Average and SD of three independent experiments . Day2 * p = 0 . 048 , Day3 * p = 0 . 036 . B ) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence ( + ) or absence ( - ) of 3 μM CH . ( C ) Assay of FoxA2 mRNA by RT - PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . * * p = 5x10 - 7 . ( D ) ChiP - seq data from a compendium of transcription factor ChIP - seq analyses ( Sánchez - Castillo et al , 2015 ) . Gene tracks represent binding of Tcf7l1 at the FoxA2 , Nodal and Eomesodermin ( Eomes ) gene locus . Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1 . ( E ) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig . 2D . Analysis was performed in wild - type ES cells in 2i culture conditions . Average and SD of three independent experiments . Klf2 * p = 0 . 025 , FoxA2 * p = 0 . 050 , Eomes * p = 0 . 023 . ( F ) ChIP for Tcf7l1 followed by qPCR . Analysis was performed in wild - type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . FoxA2 * p = 0 . 049 .

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Figure 2(A) Assay of Tcf7l1 mRNA by RT-PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH. Average and SD of three independent experiments. Day2 *p= 0.048, Day3 *p=0.036.B) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence (+) or absence (-) of 3 μM CH.(C) Assay of FoxA2 mRNA by RT-PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. **p=5x10-7.(D) ChiP-seq data from a compendium of transcription factor ChIP-seq analyses (Sánchez-Castillo et al, 2015). Gene tracks represent binding of Tcf7l1 at the FoxA2, Nodal and Eomesodermin (Eomes) gene locus. Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1.(E) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig. 2D. Analysis was performed in wild-type ES cells in 2i culture conditions. Average and SD of three independent experiments. Klf2 *p=0.025, FoxA2 *p=0.050, Eomes *p=0.023.(F) ChIP for Tcf7l1 followed by qPCR. Analysis was performed in wild-type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. FoxA2 *p = 0.049.


Figure 3 ( A ) Assay of FoxA2 mRNA by RT - PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH . Average and SD of three independent experiments . ( B ) Gene expression analysis of endodermal associated genes by RT - PCR in control wild - type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments . ( C ) FoxA2 ( green ) and Oct4 ( red ) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH . DAPI staining in blue . ( D ) Assay of CXCR4 , Hex and Sox17 mRNA by RT - PCR in control wild - type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments .

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Figure 3(A) Assay of FoxA2 mRNA by RT-PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH. Average and SD of three independent experiments.(B) Gene expression analysis of endodermal associated genes by RT-PCR in control wild-type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.(C) FoxA2 (green) and Oct4 (red) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH. DAPI staining in blue.(D) Assay of CXCR4, Hex and Sox17 mRNA by RT-PCR in control wild-type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.


Figure 4 ( A ) Assay of FoxA2 mRNA by RT - PCR in FoxA2 - Tet ES cell lines 48 hr after treatment with the indicated dose of DOX . Average and SD of two independent experiments from 2 independent lines . B ) FoxA2 immunostaining of FoxA2 - Tet ES cells 48 hr after DOX treatment . Left , no DOX control . Middle and right , 10ng / ml DOX . DAPI staining shown in blue . Right panel shows middle image without DAPI signal . ( C ) Assay of FoxA2 and CXCR4 mRNA by RT - PCR on day 3 of differentiation with DOX at the indicated dose . Average and SD of two experiments from two independent cell lines . ( D ) Assay of Sox17 mRNA by RT - PCR on day 7 of differentiation with addition of 3 µM CH and / or 10 ng / ml DOX at the indicated time points ( in days ) . Average and SD of two independent experiments from two independent cell lines . ( E ) Sox17 immunostaining of FoxA2 - Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng / ml DOX and the presence and absence of 3 µM CH as indicated . Far right panel shows image to its left without DAPI .

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Figure 4(A) Assay of FoxA2 mRNA by RT-PCR in FoxA2-Tet ES cell lines 48 hr after treatment with the indicated dose of DOX. Average and SD of two independent experiments from 2 independent lines.B) FoxA2 immunostaining of FoxA2-Tet ES cells 48 hr after DOX treatment. Left, no DOX control. Middle and right, 10ng/ml DOX. DAPI staining shown in blue. Right panel shows middle image without DAPI signal.(C) Assay of FoxA2 and CXCR4 mRNA by RT-PCR on day 3 of differentiation with DOX at the indicated dose. Average and SD of two experiments from two independent cell lines.(D) Assay of Sox17 mRNA by RT-PCR on day 7 of differentiation with addition of 3 µM CH and/or 10 ng/ml DOX at the indicated time points (in days). Average and SD of two independent experiments from two independent cell lines.(E) Sox17 immunostaining of FoxA2-Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng/ml DOX and the presence and absence of 3 µM CH as indicated. Far right panel shows image to its left without DAPI.


Figure 5 ( A ) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng / ml ( W100 ) or 200 ng / ml ( W200 ) recombinant Wnt3a plus 500 ng / ml recombinant RSpondin1 . Relative luciferase units are normalised to FOPflash . Average and SD of three independent experiments . ( B ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a ( 200 ng / ml plus 500 ng / ml RSpondin1 ( WR ) ) . Average and SD of three independent experiments . * * p < 0 . 01 . ( C ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH . Average and SD of three independent experiments . ( D ) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH . Results from two independent , clonally - derived Ctnnb1 null ES cell lines are shown . Tubulin was used as a loading control .

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Figure 5(A) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng/ml (W100) or 200 ng/ml (W200) recombinant Wnt3a plus 500 ng/ml recombinant RSpondin1. Relative luciferase units are normalised to FOPflash. Average and SD of three independent experiments.(B) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a (200 ng/ml plus 500 ng/ml RSpondin1(WR)). Average and SD of three independent experiments. ** p<0.01.(C) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH. Average and SD of three independent experiments.(D) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH. Results from two independent, clonally-derived Ctnnb1 null ES cell lines are shown. Tubulin was used as a loading control.


Figure 6 ( A ) Assay of cMyc mRNA by RT - PCR in wild - type ES cells at day 1 to 3 of differentiation . Average and SD of three independent experiments . ( B ) Immunoblot for total cMyc and phospho Ser62 ( pSer62 ) cMyc during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH . C , D ) Immunoblot for the destabilized form of cMyc protein ( pThr58 ) during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH in wild - type and Ctnnb1 null cells respectively . C , D ) Immunoblot for the destabilized form of cMyc protein ( pThr58 ) during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH in wild - type and Ctnnb1 null cells respectively . ( E ) Assay of Tcf7l1 mRNA by RT - PCR in wild - type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc ( cMyc Inhibitor II ; Myci ) . Treatment with Myc inhibitor 10058 - F4 ( Sigma ) generated similar results ( data not shown ) . Average and SD of three independent experiments . * * p = 0 . 0002 . ( F ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in cMyc null / nMyc heterozygous cells ( C - / - ; N + / - ) and floxed parental cells ( cMycfl / fl ; nMycfl / fl ( control ) ) at day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of four or six independent experiments . Tcf7l1 * p = 0 . 01 , FoxA2 * p = 0 . 034 . ( G ) FoxA2 immunostaining of control and cMyc null / nMyc heterozygous ( cMyc - / - ; nMyc + / - ) cells at day 3 of differentiation in the presence of 3 µM CH . ( H ) Assay of cMyc and Tcf7l1 mRNA by RT - PCR in cMycT58A inducible cell lines ( icMycT58A ) 24 hr after DOX treatment . Average of 2 independent experiments . ( I ) Assay of Tcf7l1 mRNA by RT - PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng / ml DOX . Average and SD of 3 independent experiments . * * p = 0 . 007 . ( J ) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng / ml .

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Figure 6(A) Assay of cMyc mRNA by RT-PCR in wild-type ES cells at day 1 to 3 of differentiation. Average and SD of three independent experiments.(B) Immunoblot for total cMyc and phospho Ser62 (pSer62) cMyc during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH.C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.(E) Assay of Tcf7l1 mRNA by RT-PCR in wild-type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc (cMyc Inhibitor II; Myci). Treatment with Myc inhibitor 10058-F4 (Sigma) generated similar results (data not shown). Average and SD of three independent experiments. **p=0.0002.(F) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in cMyc null/nMyc heterozygous cells (C-/-;N+/-) and floxed parental cells (cMycfl/fl;nMycfl/fl (control)) at day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of four or six independent experiments. Tcf7l1 *p=0.01, FoxA2 *p=0.034.(G) FoxA2 immunostaining of control and cMyc null/nMyc heterozygous (cMyc-/-;nMyc+/-) cells at day 3 of differentiation in the presence of 3 µM CH.(H) Assay of cMyc and Tcf7l1 mRNA by RT-PCR in cMycT58A inducible cell lines (icMycT58A) 24 hr after DOX treatment. Average of 2 independent experiments.(I) Assay of Tcf7l1 mRNA by RT-PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng/ml DOX. Average and SD of 3 independent experiments. **p=0.007.(J) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng/ml.


Figure 7 ( B ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the absence or presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * p = 0 . 028 . ( C ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058 - F4 . Average and SD for three independent experiments , * p = 0 . 026 . ( D ) Assay for Miz1 mRNA by RT - PCR for the first three days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . ( E ) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * * p = 0 . 0015 .

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Figure 7(B) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the absence or presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, *p=0.028.(C) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058-F4. Average and SD for three independent experiments, *p=0.026.(D) Assay for Miz1 mRNA by RT-PCR for the first three days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments.(E) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, **p=0.0015.


Figure 1B Trans - activation activity assays of CDF4 in Arabidopsis protoplasts . The full - length ORF of CDF4 and its four deletion constructs Dof ( gray boxes ) represent the DNA - binding domains of CDF4 . Negative control was the effector vector without gene inserts . The effector vector used is shown in the left panel . The GAL4 transient expression assays were performed using Arabidopsis protoplasts , shown in the right panel . Deletion of the CDF4 N - terminal domain compromised the transcriptional repression by CDF4 in our assay . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test . C Localized expression of CDF4 in a senescing rosette leaf . The ~ 50 % senesced rosette leaf was cut into three parts as indicated by the red lines . The level of CDF4 mRNA was determined in the three sections of the senescing leaf . Base ; Middle ; Tip . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . D CDF4 expression level at various developmental stages of rosette leaves . YL , young leaf ; NS , fully expanded , non - senescent leaf ; ES , early - senescent leaf , with 1 / 4 leaf area turned yellow ; LS , late - senescent leaf , with > 50 % leaf area turned yellow . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . E Effect of plant growth hormones ( 1 mM SA , 1 μM ABA , 10 μM JA and 10 μM 1 - aminocyclopropane - 1 - carboxylic acid ( ACC ) on CDF4 expression . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . F Effects of abiotic stress treatments on CDF4 expression . Rosette leaves were treated with salt ( 100 mM NaCl ) , drought , H2O2 ( 2mM ) or darkness . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . G Effects of SA , H2O2 , and dark treatment on CDF4 expression in the aba2 - 1 mutant background . AtACT2 was used as an internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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

Figure 1B Trans-activation activity assays of CDF4 in Arabidopsis protoplasts. The full-length ORF of CDF4 and its four deletion constructs Dof (gray boxes) represent the DNA-binding domains of CDF4. Negative control was the effector vector without gene inserts. The effector vector used is shown in the left panel. The GAL4 transient expression assays were performed using Arabidopsis protoplasts, shown in the right panel. Deletion of the CDF4 N-terminal domain compromised the transcriptional repression by CDF4 in our assay. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.C Localized expression of CDF4 in a senescing rosette leaf. The ~50% senesced rosette leaf was cut into three parts as indicated by the red lines. The level of CDF4 mRNA was determined in the three sections of the senescing leaf. Base; Middle; Tip. Three independent experiments were conducted. Values are given as mean ± SD, n=3.D CDF4 expression level at various developmental stages of rosette leaves. YL, young leaf; NS, fully expanded, non-senescent leaf; ES, early-senescent leaf, with 1/4 leaf area turned yellow; LS, late-senescent leaf, with >50% leaf area turned yellow. Three independent experiments were conducted. Values are given as mean ± SD, n=3.E Effect of plant growth hormones (1 mM SA, 1 μM ABA, 10 μM JA and 10 μM 1-aminocyclopropane -1-carboxylic acid (ACC) on CDF4 expression. Three independent experiments were conducted. Values are given as mean ± SD, n=3.F Effects of abiotic stress treatments on CDF4 expression. Rosette leaves were treated with salt (100 mM NaCl), drought, H2O2 (2mM) or darkness. Three independent experiments were conducted. Values are given as mean ± SD, n=3.G Effects of SA, H2O2, and dark treatment on CDF4 expression in the aba2-1 mutant background. AtACT2 was used as an internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


Figure 2A The age - dependent leaf senescence symptoms in the vector control and 35S : : CDF4 lines grown under long - day conditions were shown at 21 - days - old . The 35S : : CDF4 plants had a small leaf size and extremely early leaf senescent phenotype , while the control vector leaves were fully green . Scale bars indicate 1 cm . B The increase in SAG12 , SAG13 , PR1 and PR5 gene expression levels were investigated in 35S : : CDF4 plants . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 in comparison with the vector control by Student ' s t test . C Phenotypes of vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 plants , after treatment with 20 µM estradiol for seven days at 18 - days - old . Scale bars indicate 1 . 5 cm . E Analysis of the relative chlorophyll concentration in the sixth and seventh rosette leaves from the vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 lines shown at various development stages . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test . D The leaf senescence phenotypes of detached rosette leaves in vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 lines before and after treatment with 2 mΜ H2O2 in the dark for four days . Scale bars indicate 0 . 7 cm . F The chlorophyll concentration in leaves was analyzed . The percentage indicates the chlorophyll content relative to vector control before treatment . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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

Figure 2A The age-dependent leaf senescence symptoms in the vector control and 35S::CDF4 lines grown under long-day conditions were shown at 21-days-old. The 35S::CDF4 plants had a small leaf size and extremely early leaf senescent phenotype, while the control vector leaves were fully green. Scale bars indicate 1 cm.B The increase in SAG12, SAG13, PR1 and PR5 gene expression levels were investigated in 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 in comparison with the vector control by Student's t test.C Phenotypes of vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 plants, after treatment with 20 µM estradiol for seven days at 18-days-old. Scale bars indicate 1.5 cm.E Analysis of the relative chlorophyll concentration in the sixth and seventh rosette leaves from the vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 lines shown at various development stages. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.D The leaf senescence phenotypes of detached rosette leaves in vector control, pER8::CDF4, pER8::CDF4 -RNAi and pER8::amiCDF4 lines before and after treatment with 2 mΜ H2O2 in the dark for four days. Scale bars indicate 0.7 cm.F The chlorophyll concentration in leaves was analyzed. The percentage indicates the chlorophyll content relative to vector control before treatment. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


Figure 3A Measurement of free ABA levels in the third and fourth rosette leaves from 21 - day - old transgenic lines with altered CDF4 expression after estradiol induction for three days ( once every day ) . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test . B , C qPCR analysis of ABA synthesis and signaling - related genes in Col - 0 and 35S : : CDF4 plants . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test . D , E Relative expression of NCED2 and NCED3 in 14 - day - old CDF4GR transgenic plants treated with 20 μM β - estradiol or mock treatment for 0 , 2 , or 4 h . The expression of the corresponding genes in mock - treated plants was set to 1 . 0 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test . F , G Relative expression level of NCED2 and NCED3 in 14 - day - old CDF4GR transgenic plants treated with 20 μM DEX , 100μM CHX , DEX plus 100μM CHX , or mock . The gene expression in mock - treated plants was set to 1 . 0 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test . H Schematic diagram indicating the locations of six DOF motif clusters ( D1 to D6 ) in the NCED2 and NCED3 gene promoters . I ChIP - qPCR analysis of the ability of CDF4 to bind to the promoters of NCED2 and NCED3 . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from three - week - old pER8 : : CDF4 - HA transgenic plants . Black bars indicate the enrichment fold - changes normalized to that of ACT2 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test . Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CDF4 promoter and the p62 - SK construct with or without the CDF4 coding region and CDF4 - VP16 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activities were measured after culturing the protoplasts under low light conditions for 16 h . The ProNCED2 / 3 : LUC / Pro35S : REN ratio represents the relative activities of NCED2 and NCED3 transcription . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test . K Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CDF4 promoter and the p62 - SK construct with or without the CDF4 coding region and CDF4 - VP16 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activities were measured after culturing the protoplasts under low light conditions for 16 h . The ProNCED2 / 3 : LUC / Pro35S : REN ratio represents the relative activities of NCED2 and NCED3 transcription . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test .

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

Figure 3A Measurement of free ABA levels in the third and fourth rosette leaves from 21-day-old transgenic lines with altered CDF4 expression after estradiol induction for three days (once every day). Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.B, C qPCR analysis of ABA synthesis and signaling-related genes in Col-0 and 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.D, E Relative expression of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM β-estradiol or mock treatment for 0, 2, or 4 h. The expression of the corresponding genes in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.F, G Relative expression level of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM DEX, 100μM CHX, DEX plus 100μM CHX, or mock. The gene expression in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.H Schematic diagram indicating the locations of six DOF motif clusters (D1 to D6) in the NCED2 and NCED3 gene promoters. I ChIP-qPCR analysis of the ability of CDF4 to bind to the promoters of NCED2 and NCED3. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from three-week-old pER8::CDF4-HA transgenic plants. Black bars indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test. Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.K Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.


Figure 4A - D Transcript levels were determined by qPCR and normalized to AtACT2 . ( A , C ) Localized NCED2 and NCED3 expression in the wild - type senescing leaf . Half - senesced leaves were split into three parts , as shown in Fig . 1A , C . ( B , D ) NCED2 and NCED3 expression during leaf senescence in the vector control and CDF4 - RNAi - 1 plants . Transgenic plant leaves were analyzed from 7 to 56 d ( d , days after planting ) . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test . E Observation of rosette leaf longevity from the 26 - day - old vector control , pER8 : : CDF4 , pER8 : : CDF4 & nced2 , pER8 : : CDF4 & nced3 and pER8 : : CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks . Phenotype of detached rosette leaves arranged from oldest to youngest . Scale bars indicate 1 . 5 cm . ( F ) SAG12 expression level in rosette leaves at various development stages . Three independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test . ( G ) Chlorophyll concentration in rosette leaves at various development stages . Three independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test .

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

Figure 4A-D Transcript levels were determined by qPCR and normalized to AtACT2. (A, C) Localized NCED2 and NCED3 expression in the wild-type senescing leaf. Half-senesced leaves were split into three parts, as shown in Fig. 1A, C. (B, D) NCED2 and NCED3 expression during leaf senescence in the vector control and CDF4- RNAi-1 plants. Transgenic plant leaves were analyzed from 7 to 56 d (d, days after planting). Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.E Observation of rosette leaf longevity from the 26-day-old vector control, pER8::CDF4, pER8::CDF4 & nced2, pER8::CDF4 & nced3 and pER8::CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks. Phenotype of detached rosette leaves arranged from oldest to youngest. Scale bars indicate 1.5 cm.(F) SAG12 expression level in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.(G) Chlorophyll concentration in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.


Figure 5A The senescence phenotype of the vector control , pER8 : : CDF4 and pER8 : : CDF4 : : RNAi detached leaves treated with H2O2 and incubated in the dark for three days . The third leaf in rosettes of 3 - week - old plants was detached and incubated in MES buffer ( 2 mM MES , pH 5 . 8 ) treated with 2 mM H2O2 and 20 μM β - estradiol under dark conditions for three days . Picture shows the leaves before and after treatment . Scale bars indicate 1 cm . ( B ) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β - estradiol treatment under dark conditions for two days . The brown color represents H2O2 accumulation . Scale bars indicate 1 cm . ( C ) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β - estradiol treatment under dark conditions for two days . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test . D Chlorophyll concentrations in the leaves shown in A . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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

Figure 5A The senescence phenotype of the vector control, pER8::CDF4 and pER8::CDF4::RNAi detached leaves treated with H2O2 and incubated in the dark for three days. The third leaf in rosettes of 3-week-old plants was detached and incubated in MES buffer (2 mM MES, pH 5.8) treated with 2 mM H2O2 and 20 μM β-estradiol under dark conditions for three days. Picture shows the leaves before and after treatment. Scale bars indicate 1 cm.(B) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. The brown color represents H2O2 accumulation. Scale bars indicate 1 cm.(C) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.D Chlorophyll concentrations in the leaves shown in A. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


Figure 6A Measurement of catalase activity in vector control and CDF4RNAi plants . Ten - day - old green seedlings ( " Young " ) and the third and fourth rosette leaves from 36 - day - old plants ( " Old " ) were used , respectively . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 . B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36 - day - old plants . The expression of CAT genes in the wild - type plant is given as 1 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 . D ChIP - qPCR analysis of the relative binding of CDF4 to the promoter of CAT2 . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from 32 - d - old pER8 : : CDF4 - HA transgenic plants . Black columns indicate the enrichment fold - changes normalized to that of ACT2 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 . E Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CAT2 promoter and the p62 - SK construct with or without CDF4 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activity were measured after culturing the protoplasts under low light conditions for 16 h . six independent experiments were conducted . Values are given as mean ± SD , n = 6 . * p < 0 . 05 by Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 6A Measurement of catalase activity in vector control and CDF4RNAi plants. Ten-day-old green seedlings ("Young") and the third and fourth rosette leaves from 36-day-old plants ("Old") were used, respectively. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36-day-old plants. The expression of CAT genes in the wild-type plant is given as 1. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.D ChIP-qPCR analysis of the relative binding of CDF4 to the promoter of CAT2. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 32-d-old pER8::CDF4-HA transgenic plants. Black columns indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.E Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CAT2 promoter and the p62-SK construct with or without CDF4 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activity were measured after culturing the protoplasts under low light conditions for 16 h. six independent experiments were conducted. Values are given as mean ± SD, n=6. *p<0.05 by Student's t test.