Figure 4 Expression of the SnRK2s in yeast revealed ABF2 - mediated transactivation by SnRK2 . 1 , SnRK2 . 4 , SnRK2 . 5 , and SnRK2 . 10 in addition to OST1 . Inset : Immuno - detection of FLAG - tagged SnRK2s expressed in yeast . The data in ( B ) are normalized to control samples expressing ABF2 but no SnRK2 The active subgroup I members together with SnRK2 . 2 and SnRK2 . 3 were analyzed for differential activation of ABFs . The OST1 data shown in Fig . 1A is included for better comparison . The induction level of ABF4 relative to the empty vector control ( ev ) is indicated . Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast . Phosphorylation occurred at serine 175 or threonine 176 , and at threonine 179 . The relative abundance is given as the ratio of signal intensity of phosphorylated versus non - phosphorylated peptide for OST1 , SnRK2 . 3 , and SnRK2 . 4 in the mass spectrometric analysis . The numbering of phosphosites corresponds to OST1 . Inhibition of OST1 and SnRK2 . 4 by clade A PP2Cs . The data in ( G ) to cells expressing inactive ABI1D177A instead of ABI1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Expression of the SnRK2s in yeast revealed ABF2-mediated transactivation by SnRK2.1, SnRK2.4, SnRK2.5, and SnRK2.10 in addition to OST1. Inset: Immuno-detection of FLAG-tagged SnRK2s expressed in yeast. The data in (B) are normalized to control samples expressing ABF2 but no SnRK2  The active subgroup I members together with SnRK2.2 and SnRK2.3 were analyzed for differential activation of ABFs. The OST1 data shown in Fig. 1A is included for better comparison. The induction level of ABF4 relative to the empty vector control (ev) is indicated.  Analysis of the phosphorylation status in the activation loop of SnRK2s expressed in yeast. Phosphorylation occurred at serine 175 or threonine 176, and at threonine 179. The relative abundance is given as the ratio of signal intensity of phosphorylated versus non-phosphorylated peptide for OST1, SnRK2.3, and SnRK2.4 in the mass spectrometric analysis. The numbering of phosphosites corresponds to OST1.  Inhibition of OST1 and SnRK2.4 by clade A PP2Cs. The data in (G) to cells expressing inactive ABI1D177A instead of ABI1.


Figure 5A , B ) ABF2 - mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1 , and varying exogenous ABA concentrations . Data information : The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines . represent Replicates were normalized to controls without SnRK2 in ( A , B ) C ) Maximum ABA response recovery of samples shown in ( A , B ) relative to the reporter activity of 0 . 1 mM ABA - treated yeast cells expressing SnRK2s but no ABI1 . Data information : Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in ( C ) . D - F ) Analysis of the ABA response mediated by OST1 and SnRK2 . 4 using the receptors RCAR1 ( D ) , RCAR8 ( E ) , and RCAR14 ( F ) . Data information : The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines . The data in ( D - F ) are the mean ± s . e . m ; n = 12 , biological replicates from four independent yeast transformants for OST1 + RCAR1 / RCAR8 , and SnRK2 . 4 + RCAR8 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,B) ABF2-mediated reporter induction by different SnRK2s was assessed in the presence of the receptor complex RCAR11 and ABI1, and varying exogenous ABA concentrations. Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. represent Replicates were normalized to controls without SnRK2 in (A, B) C) Maximum ABA response recovery of samples shown in (A,B) relative to the reporter activity of 0.1 mM ABA-treated yeast cells expressing SnRK2s but no ABI1. Data information: Replicates were normalized to controls without ABI1 and RCAR for each SnRK2 in (C).  D-F) Analysis of the ABA response mediated by OST1 and SnRK2.4 using the receptors RCAR1 (D), RCAR8 (E), and RCAR14 (F). Data information: The ABA concentration that provides half maximum response recovery is indicated by dashed vertical lines. The data in (D-F) are the mean ± s.e.m; n = 12, biological replicates from four independent yeast transformants for OST1 + RCAR1/RCAR8, and SnRK2.4 + RCAR8.


Figure 6FRET - FLIM analysis of GFP : OST1 or GFP : SnRK2 . 4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA . GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana . Representative false colour images of GFP : SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2 . 4 in combination with mCherry : ABI1 and RCAR1 without exogenous ABA . The bar marks 20 µm and the nucleus ( n ) and cytosol ( c ) are indicated . Analysis of SnRK2 - ABI1 interaction as mentioned in ( A ) but in the presence of RCAR14 and different exogenous ABA levels . RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1 . Representative GFP lifetime false colour image of the analysis shown in C ) 2h after 30 µM exogenous ABA administration . Details are as described in ( B ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6FRET-FLIM analysis of GFP:OST1 or GFP:SnRK2.4 and the effect of ABI1 fused to the mCherry fluorophore in the presence of RCAR1 and ABA. GFP lifetimes were determined after transient expression of the Arabidopsis proteins in leaves of Nicotiana benthamiana.   Representative false colour images of GFP:SnRK2 lifetimes measured in leaf tissues expressing OST1 or SnRK2.4 in combination with mCherry:ABI1 and RCAR1 without exogenous ABA. The bar marks 20 µm and the nucleus (n) and cytosol (c) are indicated.    Analysis of SnRK2-ABI1 interaction as mentioned in (A) but in the presence of RCAR14 and different exogenous ABA levels. RCAR14 requires ABA for efficient displacement of SnRK2s from ABI1.   Representative GFP lifetime false colour image of the analysis shown in C) 2h after 30 µM exogenous ABA administration. Details are as described in (B).


Figure 1 ( A ) CD127 + and CD127 - cells from Lin - CD56 - population after gating on lymphoid , singlet , live , CD45 + cells of PBMCs . Lineage ( Lin ) markers include antibodies against : CD3 , CD4 , TCRαβ , TCRγδ , CD19 , CD20 , CD22 , CD34 , FcεRIα , CD11c , CD303 , CD123 , CD1a , and CD14 . ( C ) Normalized counts of Lin - CD56 - CD127 + ( red ) and Lin - CD56 - CD127 - ( blue ) cells related genes from ( B ) by DESeq2 ( n = 5 ) .

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Figure 1(A) CD127+ and CD127- cells from Lin-CD56- population after gating on lymphoid, singlet, live, CD45+ cells of PBMCs. Lineage (Lin) markers include antibodies against: CD3, CD4, TCRαβ, TCRγδ, CD19, CD20, CD22, CD34, FcεRIα, CD11c, CD303, CD123, CD1a, and CD14.(C) Normalized counts of Lin-CD56-CD127+ (red) and Lin-CD56-CD127- (blue) cells related genes from (B) by DESeq2 (n=5).


Figure 2 ( A ) Heatmap of differentially expressed genes by RNA - Seq , sorted CD56hiNK , CD56dimNK , CD56 - NK cells and ILCs , from PBMCs of 4 donors ( log2 fold change > 1 , p < 0 . 01 determined by DESeq2 ( H ) ATAC - Seq analysis of sorted CD56hiNK , CD56dimNK , CD56 - NK cells and ILCs at indicated gene loci ( representative of two donors ) .

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Figure 2(A) Heatmap of differentially expressed genes by RNA-Seq, sorted CD56hiNK, CD56dimNK, CD56-NK cells and ILCs, from PBMCs of 4 donors (log2 fold change >1, p<0.01 determined by DESeq2(H) ATAC-Seq analysis of sorted CD56hiNK, CD56dimNK, CD56-NK cells and ILCs at indicated gene loci (representative of two donors).


Figure 3C ) Representative genes for NK cell and ILC clusters . All data were generated using blood from healthy HIV - 1 - negative donors . ( G ) the expression of genes that are enriched by NK cells and / or ILCs along the pseudotime trajectory . All data were generated using blood from healthy HIV - 1 - negative donors .

O B-PANEL_START O 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

Figure 3C) Representative genes for NK cell and ILC clusters. All data were generated using blood from healthy HIV-1-negative donors.(G) the expression of genes that are enriched by NK cells and/or ILCs along the pseudotime trajectory. All data were generated using blood from healthy HIV-1-negative donors.


Figure 5 ( A ) PBMCs were stimulated with PMA and ionomycin for 3 hrs and then intracellular AREG was detected within the different subsets of NK cells and ILCs by flow cytometry . ( B ) PBMCs were cultured in MACS NK medium at 37 ° C in 5 % CO2 , 50 ng / ml IL - 2 , 50 ng / ml IL - 15 , 10 ng / ml IL - 12 or 50 ng / ml IL - 18 for 5 days , then AREG , IFN - γ or TNF - α from different subsets of NK cells and ILCs were detected . ( F PBMCs were treated with CHIR99021 ( 10 uM ) in RPMI 1640 at 37 ° C in 5 % CO2 for 48 hrs , then stimulated with PMA + ionomycin . The percentage of AREG + NK cells ( F ) are shown . two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors . G ) PBMCs were treated with CHIR99021 ( 10 uM ) in RPMI 1640 at 37 ° C in 5 % CO2 for 48 hrs , then stimulated with PMA + ionomycin . The percentage of ILCs ( G ) ( n = 15 ) are shown . two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors . ( H ) As in ( F ) , PBMCs were additionally treated with CHIR99021 and TGF - β1 ( 50 ng / ml ) before AREG + NK cells were detected by flow cytometry ( n = 8 ) . two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors . ( I ) AREG + NK cells from control or RUNX3 knockout groups were detected after IL - 12 + IL - 15 + IL - 18 stimulation in MACS NK medium at 37 ° C in 5 % CO2 for 16 hrs ( n = two - tailed paired t - test . All data were generated using blood from healthy HIV - 1 - negative donors .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 5(A) PBMCs were stimulated with PMA and ionomycin for 3 hrs and then intracellular AREG was detected within the different subsets of NK cells and ILCs by flow cytometry.(B) PBMCs were cultured in MACS NK medium at 37 °C in 5% CO2, 50 ng/ml IL-2, 50 ng/ml IL-15, 10 ng/ml IL-12 or 50 ng/ml IL-18 for 5 days, then AREG, IFN-γ or TNF-α from different subsets of NK cells and ILCs were detected.(F PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of AREG+NK cells (F) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.G) PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of ILCs (G) (n=15) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.(H) As in (F), PBMCs were additionally treated with CHIR99021 and TGF-β1 (50 ng/ml) before AREG+NK cells were detected by flow cytometry (n=8). two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.(I) AREG+NK cells from control or RUNX3 knockout groups were detected after IL-12+IL-15+IL-18 stimulation in MACS NK medium at 37°C in 5% CO2 for 16 hrs (n= two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.


Figure 6 ( A PBMCs from HIV - 1 - ( n = 20 ) , HIV - 1 + viremic ( n = 20 ) , HIV - 1 + ART suppressed ( n = 19 ) , and HIV - 1 + spontaneous controllers ( n = 20 ) were stimulated with PMA and ionomycin in RPMI 1640 at 37 ° C in 5 % CO2 for 3 hrs . The percentage ( A ) of AREG + cells from are shown for the indicated populations . Mann - Whitney test . ns , not significant . ( C - F ) Correlation of the percentage of AREG + cells in CD56 - ( C ) , CD56dim ( D ) , CD56hi ( E ) or total NK cells ( F ) , from ( A ) , with the percentage of ILCs . Spearman

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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 PBMCs from HIV-1- (n=20), HIV-1+ viremic (n=20), HIV-1+ ART suppressed (n=19), and HIV-1+ spontaneous controllers (n=20) were stimulated with PMA and ionomycin in RPMI 1640 at 37 °C in 5% CO2 for 3 hrs. The percentage (A) of AREG+ cells from are shown for the indicated populations. Mann-Whitney test. ns, not significant.(C-F) Correlation of the percentage of AREG+ cells in CD56- (C), CD56dim (D), CD56hi (E) or total NK cells (F), from (A), with the percentage of ILCs. Spearman correlation


Figure 7 ( A ) Percentage of ILCs among Lin - CD56 - cells from HIV - 1 - negative ( n = 40 ) , HIV - 1 + viremic ( n = 40 ) , HIV - 1 + ART suppressed ( n = 37 ) , and HIV - 1 + spontaneous controllers ( n = 40 ) . ( E ) Percentage of CD56 - NK cells among total NK cells from PBMCs and CD4 - PBMCs after culture in RPMI 1640 at 37 ° C in 5 % CO2 for 5 days ( n = 7 ) . ( F ) Percentage of CD56 - NK cells bearing the indicated proteins by flow cytometry , after detection in fresh PBMCs or after 5 days of culturing CD4 + T cell - depleted PBMCs ( n = 4 ) . ( G ) CD4 + T cell number ( counts / mm3 ) from the indicated groups ( n = 40 for each 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 O B-PANEL_START O O O O O O O O O O O O O O O O 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

Figure 7(A) Percentage of ILCs among Lin-CD56- cells from HIV-1-negative (n=40), HIV-1+ viremic (n=40), HIV-1+ ART suppressed (n=37), and HIV-1+ spontaneous controllers (n=40).(E) Percentage of CD56-NK cells among total NK cells from PBMCs and CD4-PBMCs after culture in RPMI 1640 at 37°C in 5% CO2 for 5 days (n=7).(F) Percentage of CD56-NK cells bearing the indicated proteins by flow cytometry, after detection in fresh PBMCs or after 5 days of culturing CD4+T cell-depleted PBMCs (n=4).(G) CD4+T cell number (counts/mm3) from the indicated groups (n=40 for each group).


Figure 8 ( A ) CD56dim and CD56 - NK cells were assessed by flow cytometry after the indicated cell populations were cultured for 5 days : total PBMCs ( n = 7 blood donors ) , PBMCs depleted of CD4 + T cells ( n = 7 blood donors ) , NK cells mixed with autologous CD4 + T cells ( n = 3 blood donors ) , and PBMCs depleted of CD4 + T cells but supplemented with IL - 2 ( n = 7 blood donors ) . ( B ) Percentage of CD56 - NK cells among total NK cells from CD4 - PBMCs cultured as in ( A ) , in the presence or absence of IL - 2 ( 10 ng / ml ) or IL - 2 neutralizing antibody ( 4 ug / ml ) ( n = 7 ) . ( C ) Percentage of CD56 - NK cells from PBMCs or CD4 - PBMCs cultured as in ( A ) in the presence or absence of IL - 2 ( 10 ng / ml ) , or IL - 15 ( 10 ng / ml ) ( n = 8 ) . ( D ) Percentage of CD56 - NK cells among total NK cells in PBMCs after culturing as in ( A ) with isotype control or IL - 2 neutralizing antibody ( 4 ug / ml ) ( n = 10 ) . ( E , F ) Percentage of CD56 - NK ( E ) or IFN - γ + NK ( F ) cells among total NK cells after culturing PBMCs as in ( A ) with TGF - β1 ( 50 ng / ml ) or TGF - β1 ( 50 ng / ml ) + IL - 2 ( 20 ng / ml ) ( n = 8 ) . ( I , J ) NSG ( n = 15 ) or human IL - 15 transgenic NSG ( NSG - huIL - 15 ) mice ( n = 15 ) were reconstituted with human CD34 + hematopoietic stem cells for 6 weeks . Blood , spleen , and liver were harvested for detection of CD56 ( I ) or IFN - γ production after 16 hrs stimulation with IL - 12 , IL - 15 , and IL - 18 ( J ) . ( K - L ) Plasma concentration of IL - 2 ( HIV - 1 - ( n = 12 ) , viremic ( n = 12 ) , ART suppressed ( n = 13 ) , controllers ( n = 14 ) ) ( K ) , TGF - β1 ( HIV - 1 - ( n = 20 ) , viremic ( n = 19 ) , ART suppressed ( n = 20 ) , controllers ( n = 20 ) ) ( L ) and IL - 15 ( HIV - 1 - ( n = 19 ) , viremic ( n = 19 ) , ART suppressed ( n = 20 ) , controllers ( n = 20 ) ) ( M ) , detected by Luminex .

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

Figure 8(A) CD56dim and CD56- NK cells were assessed by flow cytometry after the indicated cell populations were cultured for 5 days: total PBMCs (n=7 blood donors), PBMCs depleted of CD4+T cells (n=7 blood donors), NK cells mixed with autologous CD4+T cells (n=3 blood donors), and PBMCs depleted of CD4+T cells but supplemented with IL-2 (n=7 blood donors).(B) Percentage of CD56-NK cells among total NK cells from CD4-PBMCs cultured as in (A), in the presence or absence of IL-2 (10 ng/ml) or IL-2 neutralizing antibody (4 ug/ml) (n=7). (C) Percentage of CD56-NK cells from PBMCs or CD4-PBMCs cultured as in (A) in the presence or absence of IL-2 (10 ng/ml), or IL-15 (10 ng/ml) (n=8). (D) Percentage of CD56-NK cells among total NK cells in PBMCs after culturing as in (A) with isotype control or IL-2 neutralizing antibody (4 ug/ml) (n=10).(E, F) Percentage of CD56-NK (E) or IFN-γ+ NK (F) cells among total NK cells after culturing PBMCs as in (A) with TGF-β1 (50 ng/ml) or TGF-β1 (50 ng/ml) + IL-2 (20 ng/ml) (n=8).(I, J) NSG (n=15) or human IL-15 transgenic NSG (NSG-huIL-15) mice (n=15) were reconstituted with human CD34+ hematopoietic stem cells for 6 weeks. Blood, spleen, and liver were harvested for detection of CD56 (I) or IFN-γ production after 16 hrs stimulation with IL-12, IL-15, and IL-18 (J).(K-L) Plasma concentration of IL-2 (HIV-1- (n=12), viremic (n=12), ART suppressed (n=13), controllers (n=14)) (K), TGF-β1 (HIV-1- (n=20), viremic (n=19), ART suppressed (n=20), controllers (n=20)) (L) and IL-15 (HIV-1- (n=19), viremic (n=19), ART suppressed (n=20), controllers (n=20)) (M), detected by Luminex.


Figure 9 ( C ) Log2 fold change of mTOR signaling - related genes , showing cells stimulated with IL - 12 , IL - 15 , and IL - 18 versus unstimulated CD56 - or CD56dim NK cells . ns , not significant . All data were generated using blood from healthy HIV - 1 - negative donors .

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Figure 9(C) Log2 fold change of mTOR signaling-related genes, showing cells stimulated with IL-12, IL-15, and IL-18 versus unstimulated CD56- or CD56dim NK cells. ns, not significant. All data were generated using blood from healthy HIV-1-negative donors.


Figure 10 ( A ) PBMCs were cultured at 37 ° C in 5 % CO2 with or without IL - 2 ( 50 ng / ml ) for 5 days , the phosphorylation of mTOR on Ser2448 , AKT on Ser473 , 4EBP1 on Thr36 and Thr45 , and S6 on Ser235 and Ser236 , and the surface CD71 from NK cells ( Lin - TBX21 + ) were detected by flow cytometry ( n = 8 ) . ( B ) As in ( A ) , PBMCs or CD4 - PBMCs were cultured for 5 days , the indicated targets from NK cells were detected ( n = 8 ) . ( C ) As in ( A ) , PBMCs were cultured with or without IL - 15 ( 50 ng / ml ) for 5 days , the indicated targets were detected ( n = 8 ) . All data were generated using blood from healthy HIV - 1 - negative donors . ( E , F ) PBMCs or CD4 - PBMCs were cultured in the presence or absence of rapamycin ( 10 nM ) ( F , n = 8 ) or Torin 1 ( 250 nM ) ( G , n = 8 ) , with or without IL - 2 ( 10 ng / ml ) or IL - 15 ( 10 ng / ml ) , for 5 days . IFN - γ was detected in Lin - TBX21 + cells . All data were generated using blood from healthy HIV - 1 - negative donors . ( G , H ) PBMCs were cultured in the presence or absence of rapamycin ( 10 nM ) ( F ) or Torin 1 ( 250 nM ) combined with or without IL - 2 ( 10 ng / ml ) ( G , n = 8 ) or IL - 15 ( 10 ng / ml ) ( H , n = 8 ) for 5 days . The indicated targets from the Lin - TBX21 + population were detected as in ( A ) . All data were generated using blood from healthy HIV - 1 - negative donors .

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

Figure 10(A) PBMCs were cultured at 37 °C in 5% CO2 with or without IL-2 (50 ng/ml) for 5 days, the phosphorylation of mTOR on Ser2448, AKT on Ser473, 4EBP1 on Thr36 and Thr45, and S6 on Ser235 and Ser236, and the surface CD71 from NK cells (Lin-TBX21+) were detected by flow cytometry (n=8). (B) As in (A), PBMCs or CD4-PBMCs were cultured for 5 days, the indicated targets from NK cells were detected (n=8). (C) As in (A), PBMCs were cultured with or without IL-15 (50 ng/ml) for 5 days, the indicated targets were detected (n=8). All data were generated using blood from healthy HIV-1-negative donors.(E, F) PBMCs or CD4-PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F, n=8) or Torin 1 (250 nM) (G, n=8), with or without IL-2 (10 ng/ml) or IL-15 (10 ng/ml), for 5 days. IFN-γ was detected in Lin-TBX21+ cells. All data were generated using blood from healthy HIV-1-negative donors.(G, H) PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F) or Torin 1 (250 nM) combined with or without IL-2 (10 ng/ml) (G, n=8) or IL-15 (10 ng/ml) (H, n=8) for 5 days. The indicated targets from the Lin-TBX21+ population were detected as in (A). All data were generated using blood from healthy HIV-1-negative donors.


Figure 1A ) Overview of immunofluorescence staining for centrosomes ( TUBG1 ) and cilia ( ARL13B ) in control ( Con ) ( left ) and EPZ treated sections ( right ) . Dotted lines mark either ventricular ( bottom part ) or pial ( top part ) surface of the tissue . Data information : Scale bars in all panels : 10 µm . B ) Left : quantification of total centrosome numbers in control and EPZ treated sections in 100 µm . Right : number of centrosomes in ventricular zone ( VZ ) , subventricular zone ( SVZ ) , and cortical plate ( CP ) expressed as percentage of total per condition . C ) Left : quantification of total cilia numbers in control and EPZ treated sections in 100 µm . Right : number of cilia in VZ , SVZ , and CP expressed as percentage of total per condition . Data information : T - test , two - tails was performed for all quantifications , * p < 0 . 05 , * * * p < 0 . 001 , error bars represent SD . D ) Overview of VZ showing TUBG1 and ARL13B immunofluorescence staining in control ( top ) and EPZ ( bottom ) condition . Squared parentheses indicate the ventricular and abventricular compartment . Dotted lines mark ventricular surface of tissue slide . Data information : Scale bars in all panels : 10 µm . E ) Quantification of TUBG1 - and ( F ) ARL13B - positive structures in ventricular and abventricular compartment expressed as percentage of the total number per condition . Data information : T - test , two - tails was performed for all quantifications , * p < 0 . 05 , * * * p < 0 . 001 , error bars represent SD .

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

Figure 1A) Overview of immunofluorescence staining for centrosomes (TUBG1) and cilia (ARL13B) in control (Con) (left) and EPZ treated sections (right). Dotted lines mark either ventricular (bottom part) or pial (top part) surface of the tissue . Data information: Scale bars in all panels: 10 µm.B) Left: quantification of total centrosome numbers in control and EPZ treated sections in 100 µm. Right: number of centrosomes in ventricular zone (VZ), subventricular zone (SVZ), and cortical plate (CP) expressed as percentage of total per condition. C) Left: quantification of total cilia numbers in control and EPZ treated sections in 100 µm. Right: number of cilia in VZ, SVZ, and CP expressed as percentage of total per condition. Data information: T-test, two-tails was performed for all quantifications, *p<0.05, ***p<0.001, error bars represent SD.D) Overview of VZ showing TUBG1 and ARL13B immunofluorescence staining in control (top) and EPZ (bottom) condition. Squared parentheses indicate the ventricular and abventricular compartment. Dotted lines mark ventricular surface of tissue slide. Data information: Scale bars in all panels: 10 µm.E) Quantification of TUBG1- and (F) ARL13B-positive structures in ventricular and abventricular compartment expressed as percentage of the total number per condition. Data information: T-test, two-tails was performed for all quantifications, *p<0.05, ***p<0.001, error bars represent SD.


Figure 2A ) Immunostaining of control and EPZ treated sections showing SOX2 and EOMES expression and EdU labelling . Scale bar : 20 µm . Dotted lines mark ventricular surface of the tissue . B ) Quantification of SOX2 - , EOMES - , EdU - positive cells , and co - labelled cells normalised to 100 μm of apical surface . T - test was performed , * p < 0 . 05 , * * p < 0 . 01 , error bars represent SEM . Total n of independent experiments for EdU labelling : 9 ( n = 4 control samples , n = 5 EPZ - treated samples ) . Total n of independent experiments for SOX2 , EOMES , and double stainings : 6 ( n = 3 control samples , n = 3 EPZ - treated samples ) . E ) Immunofluorescence staining showing overview of CTIP2 - positive cells in control and EPZ treated sections . Scale bars : 20 µm . Dotted lines mark either ventricular or pial surface of the tissue . F ) Quantification of total CTIP2 - positive cells , normalised to 100 µm of apical surface . Total n of independent experiments : 6 ( n = 3 control samples , n = 3 EPZ - treated samples ) . Error bars represent SEM . G ) Immunofluorescence staining showing SOX2 - , EOMES - , and CTIP2 - positive cells in control and EPZ treated sections . Scale bars : 10 μm . Dotted lines mark the ventricular surface of the tissue . Magnifications on the right show exemplarily a CTIP2 positive cell ( arrowhead ) that is negative for SOX2 and positive for EOMES in EPZ treated condition . H ) Quantification of CTIP2 - positive cells in the VZ , normalised to 100 µm of apical surface . Total n of independent experiments : 6 ( n = 3 control samples , n = 3 EPZ - treated samples ) . T - test was performed , * * p < 0 . 01 , error bars represent SEM . I ) In utero electroporation of cortical tissue of shRNA against DOT1L and control alongside a GFP expressing plasmid , followed by assessment of expression of CTIP2 and TUBB3 as markers for neuronal differentiation to study cell - autonomous and non - autonomous effects . Scale bars : 20 µm . Dotted lines mark either ventricular or pial surface of tissue slide . J ) Quantification of CTIP2 - , and TUBB3 - positive cells in VZ , SVZ , IZ , and CP in control and shDot1l sections . Total n of independent experiments : 6 ( n = 3 control shRNA , n = 3 shDot1l ) . T - test was performed , * p < 0 . 05 , error bars represent SEM .

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

Figure 2A) Immunostaining of control and EPZ treated sections showing SOX2 and EOMES expression and EdU labelling. Scale bar: 20 µm. Dotted lines mark ventricular surface of the tissue. B) Quantification of SOX2-, EOMES-, EdU- positive cells, and co-labelled cells normalised to 100 μm of apical surface. T-test was performed, *p<0.05, **p<0.01, error bars represent SEM. Total n of independent experiments for EdU labelling: 9 (n=4 control samples, n=5 EPZ-treated samples). Total n of independent experiments for SOX2, EOMES, and double stainings: 6 (n=3 control samples, n=3 EPZ-treated samples).E) Immunofluorescence staining showing overview of CTIP2-positive cells in control and EPZ treated sections. Scale bars: 20 µm. Dotted lines mark either ventricular or pial surface of the tissue. F) Quantification of total CTIP2-positive cells, normalised to 100 µm of apical surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). Error bars represent SEM.G) Immunofluorescence staining showing SOX2-, EOMES-, and CTIP2-positive cells in control and EPZ treated sections. Scale bars: 10 μm. Dotted lines mark the ventricular surface of the tissue. Magnifications on the right show exemplarily a CTIP2 positive cell (arrowhead) that is negative for SOX2 and positive for EOMES in EPZ treated condition. H) Quantification of CTIP2-positive cells in the VZ, normalised to 100 µm of apical surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). T-test was performed, **p<0.01, error bars represent SEM.I) In utero electroporation of cortical tissue of shRNA against DOT1L and control alongside a GFP expressing plasmid, followed by assessment of expression of CTIP2 and TUBB3 as markers for neuronal differentiation to study cell-autonomous and non-autonomous effects. Scale bars: 20 µm. Dotted lines mark either ventricular or pial surface of tissue slide. J) Quantification of CTIP2-, and TUBB3-positive cells in VZ, SVZ, IZ, and CP in control and shDot1l sections. Total n of independent experiments: 6 (n=3 control shRNA, n=3 shDot1l ). T-test was performed, *p<0.05, error bars represent SEM.


Figure 3A ) Microinjected cells in the VZ ( magenta ) in sections treated with DMSO ( Con ) ( upper panel ) or EPZ ( lower panel ) showing Dx - A555 labelled cells after 24 h in slice culture . White arrow indicates the apical process contacting the ventricular surface . White arrowheads indicate the apical - directed process of delaminated cells . Scale bars : 10 µm . B ) Immunofluorescence staining of microinjected sections showing the progeny of microinjected cells ( a daughter cell pair ) and expression of EOMES and TUBB3 . White arrows indicate the apical - directed processes . Note that in the top panel the apical - directed process contacts the ventricular surface ( dotted white line ) ( EOMES - , TUBB3 - negative ) , while in the bottom panel it does not contact the ventricular surface , indicating that the cell has delaminated ( EOMES - , TUBB3 - positive ) . Scale bars MIP : 10 µm ; insets : 5 µm . Data information : VZ - Ventricular Zone , SVZ - Subventricular Zone , CP - Cortical Plate . Scale bars in all main panels : 20 µm , and all insets : 5 µm . C ) Quantification of microinjected cells found as single - cell or two - cells clusters , expressed as percentage of total Dx - A555 - positive cells per condition . D ) Quantification of microinjected cells with or without contact to the apical surface of the organotypic slice expressed as percentage of total Dx - A555 - positive cells per condition . Total n of independent microinjection experiments : 6 ( n = 3 wild type embryos , n = 3 in Tis21 - GFP embryos ) . Total number of cells scored : Con , 156 cells ; EPZ , 287 cells . E ) EOMES - positive and EOMES - negative cells plotted as a percentage of total cells scored per condition . F ) TUBB3 - positive and TUBB3 - negative cells in control and EPZ conditions plotted as a percentage of total cells scored per condition . Data information : Cell scoring based on TUBB3 or EOMES expression are from three independent experiments ( n = 3 ; Con : 70 cells , EPZ : 113 cells ) . Fisher ' s exact test was performed for all quantifications , * * * p < 0 . 001 , error bars represent SEM . MIP - maximum intensity projection .

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

Figure 3A) Microinjected cells in the VZ (magenta) in sections treated with DMSO (Con) (upper panel) or EPZ (lower panel) showing Dx-A555 labelled cells after 24 h in slice culture. White arrow indicates the apical process contacting the ventricular surface. White arrowheads indicate the apical-directed process of delaminated cells. Scale bars: 10 µm. B) Immunofluorescence staining of microinjected sections showing the progeny of microinjected cells (a daughter cell pair) and expression of EOMES and TUBB3. White arrows indicate the apical-directed processes. Note that in the top panel the apical-directed process contacts the ventricular surface (dotted white line) (EOMES-, TUBB3-negative), while in the bottom panel it does not contact the ventricular surface, indicating that the cell has delaminated (EOMES-, TUBB3-positive). Scale bars MIP: 10 µm; insets: 5 µm. Data information: VZ - Ventricular Zone, SVZ - Subventricular Zone, CP - Cortical Plate. Scale bars in all main panels: 20 µm, and all insets: 5 µm.C) Quantification of microinjected cells found as single-cell or two-cells clusters, expressed as percentage of total Dx-A555-positive cells per condition. D) Quantification of microinjected cells with or without contact to the apical surface of the organotypic slice expressed as percentage of total Dx-A555-positive cells per condition. Total n of independent microinjection experiments: 6 (n=3 wild type embryos, n=3 in Tis21-GFP embryos). Total number of cells scored: Con, 156 cells; EPZ, 287 cells. E) EOMES-positive and EOMES-negative cells plotted as a percentage of total cells scored per condition. F) TUBB3-positive and TUBB3-negative cells in control and EPZ conditions plotted as a percentage of total cells scored per condition. Data information: Cell scoring based on TUBB3 or EOMES expression are from three independent experiments (n=3; Con: 70 cells, EPZ: 113 cells). Fisher's exact test was performed for all quantifications, ***p<0.001, error bars represent SEM. MIP - maximum intensity projection.


Figure 4A ) Microinjected cells in the VZ ( magenta ) of control ( Con ) and EPZ treated slices 24 h after microinjection and slice culture . Left : daughter cell pair in control section with asymmetric GFP expression , with cell 1 ( basal location ) being mild GFP - positive , and cell 2 ( apical location ) GFP - negative . Right : EPZ treated section showing a daughter cell pair with symmetric mild GFP - positive expression . Scale bars : 5 µm . Dotted shapes indicate the contour of Dx - A555 cell soma . B ) Graph showing proportion of labelled cells expressing GFP - positive , - mild positive , or - negative , expressed as percentage of total cells per condition . Data information : Quantifications are based on three independent experiments ( n = 3 , biological replicates ) and Chi - square test was performed in all cases . Error bars represent SEM , * * * p < 0 . 001 , * * * * p < 0 . 0001 , alpha = 0 . 05 . VZ - ventricular zone . The following number of cells were scored : B ) TIS21 - GFP - 267 total cells were quantified ( Con : 86 cells , EPZ : 181 cells ) . C ) Symmetry or asymmetry in daughter cells ' apical contact ( yes / yes : both cells have apical contact ; yes / no : one cell has apical contact and the other not ; no / no : both cells lack apical contact ) . D ) Symmetry ( pos / pos , neg / neg ) or asymmetry ( pos / neg ) in GFP expression in daughter cells expressed as percentage of total cells per condition . E ) Symmetry or asymmetry in TUBB3 expression in daughter cells plotted ( pos / pos : both cells express TUBB3 ; pos / neg : one cell expresses TUBB3 and the other not ; neg / neg : none of the cells expresses TUBB3 ) . Data information : Quantifications are based on three independent experiments ( n = 3 , biological replicates ) and Chi - square test was performed in all cases . Error bars represent SEM , * * * p < 0 . 001 , * * * * p < 0 . 0001 , alpha = 0 . 05 . The following number of cells were scored : C ) Apical contact - 85 total daughter cell pairs ( Con : 31 pairs , EPZ : 54 pairs ) . D ) TIS21 - GFP - 58 total daughter cell pairs ( Con : 21 pairs , EPZ : 37 pairs ) . E ) TUBB3 - 50 total daughter cell pairs ( Con : 17 pairs , EPZ : 33 pairs ) . F ) Phalloidin staining of control and EPZ treated sections showing cells during mitosis . Scale bar : 5 µm . G ) Pie chart showing the proportion of cells with different angles of the cleavage plane in respect to the ventricular surface , divided in groups with increments of 30 ° . H ) Percentage of cells undergoing symmetric or asymmetric cell division , defined by the cleavage plane bypassing or bysecting the apical plasma membrane , in control and EPZ treated samples . Data information : Quantifications are based on three independent experiments ( n = 3 , biological replicates ) and Chi - square test was performed in all cases . Error bars represent SEM , * * * p < 0 . 001 , * * * * p < 0 . 0001 , alpha = 0 . 05 . VZ - ventricular zone . The following number of cells were scored : G ) For control condition 41 cells from four independent samples , 5 sections and 15 different images were assessed . EPZ treated quantifications were performed on a total of 26 cells from 3 independent samples , 5 sections and 16 images . H ) For control condition a total of 37 cells from four independent samples , 5 sections and 15 different images were assessed . EPZ treated quantifications were performed on a total of 23 cells from 3 independent samples , 5 sections and 16 images .

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

Figure 4A) Microinjected cells in the VZ (magenta) of control (Con) and EPZ treated slices 24 h after microinjection and slice culture. Left: daughter cell pair in control section with asymmetric GFP expression, with cell 1 (basal location) being mild GFP-positive, and cell 2 (apical location) GFP-negative. Right: EPZ treated section showing a daughter cell pair with symmetric mild GFP-positive expression. Scale bars: 5 µm. Dotted shapes indicate the contour of Dx-A555 cell soma. B) Graph showing proportion of labelled cells expressing GFP-positive, -mild positive, or -negative, expressed as percentage of total cells per condition. Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. VZ - ventricular zone. The following number of cells were scored: B) TIS21-GFP - 267 total cells were quantified (Con: 86 cells, EPZ: 181 cells).C) Symmetry or asymmetry in daughter cells' apical contact (yes/yes: both cells have apical contact; yes/no: one cell has apical contact and the other not; no/no: both cells lack apical contact). D) Symmetry (pos/pos, neg/neg) or asymmetry (pos/neg) in GFP expression in daughter cells expressed as percentage of total cells per condition. E) Symmetry or asymmetry in TUBB3 expression in daughter cells plotted (pos/pos: both cells express TUBB3; pos/neg: one cell expresses TUBB3 and the other not; neg/neg: none of the cells expresses TUBB3). Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. The following number of cells were scored: C) Apical contact - 85 total daughter cell pairs (Con: 31 pairs, EPZ: 54 pairs). D) TIS21-GFP - 58 total daughter cell pairs (Con: 21 pairs, EPZ: 37 pairs). E) TUBB3 - 50 total daughter cell pairs (Con: 17 pairs, EPZ: 33 pairs).F) Phalloidin staining of control and EPZ treated sections showing cells during mitosis. Scale bar: 5 µm. G) Pie chart showing the proportion of cells with different angles of the cleavage plane in respect to the ventricular surface, divided in groups with increments of 30° . H) Percentage of cells undergoing symmetric or asymmetric cell division, defined by the cleavage plane bypassing or bysecting the apical plasma membrane, in control and EPZ treated samples. Data information: Quantifications are based on three independent experiments (n=3, biological replicates) and Chi-square test was performed in all cases. Error bars represent SEM, ***p<0.001, ****p<0.0001, alpha = 0.05. VZ - ventricular zone. The following number of cells were scored: G) For control condition 41 cells from four independent samples, 5 sections and 15 different images were assessed. EPZ treated quantifications were performed on a total of 26 cells from 3 independent samples, 5 sections and 16 images. H) For control condition a total of 37 cells from four independent samples, 5 sections and 15 different images were assessed. EPZ treated quantifications were performed on a total of 23 cells from 3 independent samples, 5 sections and 16 images.


Figure 6C ) Immunostaining showing ASNS - positive cells in the VZ , SVZ and CP of control and EPZ treated sections ; and signal intensity for each condition . Scale bars : 50 µm . Dotted lines mark either ventricular or pial surface of the tissue . D ) ChIP - seq and ATAC - seq tracks showing distribution of peaks for selected chromatin marks on the Asns locus for NPCs and cerebral cortex of E12 . 5 and E14 . 5 mice . Tracks for control and EPZ conditions are compared in NPCs . Indicated is the TSS of Asns . F ) ChIP - qPCR analysis for DOT1L and IgG ( n = 7 ; biological replicates ) at the TSS on Asns genomic locus in in vitro - derived NPCs . One - sided paired Wilcoxon statistical test was performed , error bars represent SEM . * * p < 0 . 01 . G ) ChIP - qPCR analyses for EZH2 ( n = 5 ; biological replicates ) and H3K27me3 ( n = 9 ; biological replicates ) at the TSS on Asns genomic locus in control and EPZ treated condition of in vitro - derived NPCs . One - sided paired Wilcoxon statistical test was performed , error bars represent SEM . * p < 0 . 04 .

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Figure 6C) Immunostaining showing ASNS-positive cells in the VZ, SVZ and CP of control and EPZ treated sections; and signal intensity for each condition. Scale bars: 50 µm. Dotted lines mark either ventricular or pial surface of the tissue .D) ChIP-seq and ATAC-seq tracks showing distribution of peaks for selected chromatin marks on the Asns locus for NPCs and cerebral cortex of E12.5 and E14.5 mice. Tracks for control and EPZ conditions are compared in NPCs. Indicated is the TSS of Asns.F) ChIP-qPCR analysis for DOT1L and IgG (n=7; biological replicates) at the TSS on Asns genomic locus in in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. **p<0.01. G) ChIP-qPCR analyses for EZH2 (n=5; biological replicates) and H3K27me3 (n=9; biological replicates) at the TSS on Asns genomic locus in control and EPZ treated condition of in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. *p<0.04.


Figure 7C ) Ex utero electroporated cells expressing GFP ( green ) or TUBB3 ( magenta ) in control ( Con ; pCMV - GFP ) or in ASNS overexpression ( ASNS OE ; pCMV - ASNS - GFP ) condition 24 h after slice culture . Scale bars : 20 µm . Dotted lines mark ventricular surface of the tissue . Magnifications on the right show exemplarily a GFP and TUBB3 double positive cell upon ASNS overexpression . Dotted shapes indicate the border of cell soma based on GFP expression . D ) Fraction of TUBB3 - positive and - negative cells expressed as percentage of total cells in Con and upon ASNS OE . Quantifications were made from independent experiments ( n = 3 ) . Student ' s t - test , * * p < 0 . 01 , error bars represent SEM . E ) Representative images of mCherry ( red ) or TBR1 - expressing ( green ) cells in control ( Con ; pCAG - mCherry ) or in ASNS overexpression ( ASNS OE ; pCAG - ASNS ) condition , 48 h after in utero electroporation . Scale bars : 20 µm . Dotted lines mark ventricular surface of the tissue . Magnifications on the right show exemplarily mCherry and TBR1 double or single positive cells upon ASNS overexpression . F ) Fraction of TBR1 - positive and - negative cells expressed as percentage of total cells in Con and upon ASNS OE . Quantifications were made from 1141 cells in control group and 606 cells in ASNS OE group . Fisher ' s exact test , * * * * p < 0 . 0001 .

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Figure 7C) Ex utero electroporated cells expressing GFP (green) or TUBB3 (magenta) in control (Con; pCMV-GFP) or in ASNS overexpression (ASNS OE; pCMV-ASNS-GFP) condition 24 h after slice culture. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily a GFP and TUBB3 double positive cell upon ASNS overexpression. Dotted shapes indicate the border of cell soma based on GFP expression. D) Fraction of TUBB3-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from independent experiments (n=3). Student's t-test, **p<0.01, error bars represent SEM.E) Representative images of mCherry (red) or TBR1-expressing (green) cells in control (Con; pCAG-mCherry) or in ASNS overexpression (ASNS OE; pCAG-ASNS) condition, 48 h after in utero electroporation. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily mCherry and TBR1 double or single positive cells upon ASNS overexpression. F) Fraction of TBR1-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from 1141 cells in control group and 606 cells in ASNS OE group. Fisher's exact test, ****p<0.0001.


Figure 1Widefield images of RPA - mCherry - ssDNA molecules ( magenta ) in the presence of GFP - Rdh54 ( green ; 1 , 3 , 10 and 30 nM ) . Widefield images of Rad51 - ssDNA molecules ( unlabeled ) in the presence of GFP - Rdh54 ( green ; 1 , 3 , 10 and 30 nM ) . Kymograph illustrating the dissociation of GFP - Rdh54 from Rad51 - ssDNA ; arrowhead highlights the time point at which free GFP - Rdh54 was flushed from the sample chamber . Graph depicting the dissociation rate of GFP - Rdh54 from Rad51 - ssDNA . Error bars represent s . d . of individual ssDNA molecules ( N = 25 ) . Kymograph showing the association of GFP - Rdh54 with a Rad51 - ssDNA molecule ; arrowhead highlights the injection time point for GFP - Rdh54 . Association rates of GFP - Rdh54 on individual Rad51 PSCs ( N = 44 ) . Red lines represent the mean and s . d .

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Figure 1Widefield images of RPA-mCherry-ssDNA molecules (magenta) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).Widefield images of Rad51-ssDNA molecules (unlabeled) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).Kymograph illustrating the dissociation of GFP-Rdh54 from Rad51-ssDNA; arrowhead highlights the time point at which free GFP-Rdh54 was flushed from the sample chamber.   Graph depicting the dissociation rate of GFP-Rdh54 from Rad51-ssDNA. Error bars represent s.d. of individual ssDNA molecules (N=25).   Kymograph showing the association of GFP-Rdh54 with a Rad51-ssDNA molecule; arrowhead highlights the injection time point for GFP-Rdh54.   Association rates of GFP-Rdh54 on individual Rad51 PSCs (N=44). Red lines represent the mean and s.d.


Figure 2Images of Rad51 - ssDNA molecules bound by both mCherry - Rdh54 ( 30 nM ; magenta ) and GFP - Rad54 ( green ; 30 nM ) . Quantification of bound GFP - Rad54 ( 10 nM ) signal intensity for Rad51 - ssDNA molecules with and without bound mCherry - Rdh54 ( 10 nM ) . Error bars represent s . d . of individual ssDNA molecules ( N = 21 ) . Images showing increasing concentrations of GFP - Rad54 ( green ) only or mCherry - Rdh54 ( magenta ) only bound separately to Rad51 - ssDNA molecules . Images of GFP - Rad54 ( green ) and mCherry - Rdh54 ( magenta ) co - bound to the same Rad51 - ssDNA molecules . Quantification of GFP - Rad54 binding in the absence and presence of mCherry - Rdh54 . The data was fit by non - linear regression and error bars represent s . d . of individual molecules ( N = 43 ) . Quantification of mCherry - Rdh54 binding in the absence or presence of GFP - Rad54 . The data was fit by non - linear regression and error bars represent s . d . of individual molecules ( N = 43 ) .

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Figure 2Images of Rad51-ssDNA molecules bound by both mCherry-Rdh54 (30 nM; magenta) and GFP-Rad54 (green; 30 nM).Quantification of bound GFP-Rad54 (10 nM) signal intensity for Rad51-ssDNA molecules with and without bound mCherry-Rdh54 (10 nM). Error bars represent s.d. of individual ssDNA molecules (N = 21).Images showing increasing concentrations of GFP-Rad54 (green) only or mCherry-Rdh54 (magenta) only bound separately to Rad51-ssDNA molecules.Images of GFP-Rad54 (green) and mCherry-Rdh54 (magenta) co-bound to the same Rad51-ssDNA molecules.Quantification of GFP-Rad54 binding in the absence and presence of mCherry-Rdh54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).Quantification of mCherry-Rdh54 binding in the absence or presence of GFP-Rad54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).


Figure 3Images of GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 and Rad54NRdh54 ( 1 , 3 , 10 and 30 nM each ) bound to Rad51 - ssDNA molecules . Images of GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 and Rad54NRdh54 ( 1 , 3 , 10 and 30 nM each ) bound to Rad51 - ssDNA molecules in the presence of 30 nM Hed1 . Quantification of the integrated GFP signal intensity for GFP - tagged Rad54 ( N = 75 ) , Rdh54NRad54 ( N = 46 ) , Rdh54 ( N = 50 ) and Rad54NRdh54 ( N = 35 ; 1 , 3 , 10 and 30 nM each ) bound to Rad51 - ssDNA molecules . The data was fit by non - linear regression and error bars represent s . d . for individual DNA molecules . Comparison of fold binding inhibition for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 and Rad54NRdh54 in reactions conducted in the presence of 30 nM Hed1 . The dot represents the mean and the error bars represent the range for two independent experiments .

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Figure 3Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules.Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules in the presence of 30 nM Hed1.Quantification of the integrated GFP signal intensity for GFP-tagged Rad54 (N = 75), Rdh54NRad54 (N = 46), Rdh54 (N = 50) and Rad54NRdh54 (N = 35; 1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules. The data was fit by non-linear regression and error bars represent s.d. for individual DNA molecules.Comparison of fold binding inhibition for GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 in reactions conducted in the presence of 30 nM Hed1. The dot represents the mean and the error bars represent the range for two independent experiments.


Figure 4Widefield images of Atto565 - labeled PSCs ( magenta ) bound to dsDNA curtains stained with YOYO1 ( green ) . The PSCs were prepared with the indicated combinations of Rad51 , Rdh54 and GFP - Rad54 . Number of Atto565 - labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only ( minus Rad51 ; N = 372 ) , Rad51 plus Rad54 only ( N = 117 ) , Rad51 plus Rdh54 only ( N = 258 ) , Rad51 plus Rdh54 and Rad54 ( N = 303 ) . The error bars represent the s . d . for three independent experiments .

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Figure 4Widefield images of Atto565-labeled PSCs (magenta) bound to dsDNA curtains stained with YOYO1 (green). The PSCs were prepared with the indicated combinations of Rad51, Rdh54 and GFP-Rad54.   Number of Atto565-labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only (minus Rad51 ; N = 372), Rad51 plus Rad54 only (N= 117), Rad51 plus Rdh54 only (N = 258), Rad51 plus Rdh54 and Rad54 (N = 303). The error bars represent the s.d. for three independent experiments.


Figure 5Kymographs of Rad51 - tailed duplex PSCs ( magenta ) containing the indicated combinations of Rad54 and Rdh54 proteins . Translocation velocities for tailed duplex PSCs prepared with Rad54 only ( lane 1 ; N = 86 ) , Rad54 plus Rdh54 ( lane 2 ; N = 144 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 47 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 112 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; N = 64 ) . The error bars represent s . d . Translocation distances for tailed duplex PSCs prepared with Rad54 only ( lane 1 ; N = 86 ) , Rad54 plus Rdh54 ( lane 2 ; N = 144 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 47 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 112 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; N = 64 ) . Error bars were generated by bootstrapping . First passage recognition efficiency for tailed duplex PSCs prepared with Rad54 only ( lane 1 ; N = 145 ) , Rad54 plus Rdh54 ( lane 2 ; N = 115 ) , Rad54 - K341R plus Rdh54 ( lane 3 ; N = 45 ) , Rad54 plus Rdh54 - K318R ( lane 4 ; N = 39 ) , or Rad54 - K341R plus Rdh54 - K318R ( lane 5 ; no translocation observed ) . Error bars ( s . d . ) were generated from three independent experiments . PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 ( N = 181 ) ; Rad54 plus Rdh54 - K318R ( N = 115 ) ; Rad54 - K341R plus Rdh54 ( N = 90 ) ; or Rad54 - K341R plus Rdh54 - K318R ( N = 54 ) , as indicated . Error bars were generated by bootstrapping . The asterisks denote the targeted region of homology on the donor dsDNA molecules .

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Figure 5Kymographs of Rad51-tailed duplex PSCs (magenta) containing the indicated combinations of Rad54 and Rdh54 proteins.Translocation velocities for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). The error bars represent s.d.   Translocation distances for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). Error bars were generated by bootstrapping.   First passage recognition efficiency for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 145), Rad54 plus Rdh54 (lane 2; N = 115), Rad54-K341R plus Rdh54 (lane 3; N = 45), Rad54 plus Rdh54-K318R (lane 4; N = 39), or Rad54-K341R plus Rdh54-K318R (lane 5; no translocation observed). Error bars (s.d.) were generated from three independent experiments.   PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 (N= 181); Rad54 plus Rdh54-K318R (N= 115); Rad54-K341R plus Rdh54 (N = 90); or Rad54-K341R plus Rdh54-K318R (N = 54), as indicated. Error bars were generated by bootstrapping. The asterisks denote the targeted region of homology on the donor dsDNA molecules.


Figure 6D - loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 ( 300 nM ) , tailed duplex substrate ( 15 nM ) and a pUC19 plasmid dsDNA ( 0 . 3 nM ) . Quantification of D - loop formation for Rad54 and Rdh54NRad54 . Error bars represent s . d . of three independent experiments . D - loop stimulation by Rdh54 and Rad54NRdh54 . Quantification of D - loop formation for Rad54 and Rdh54NRad54 . Error bars represent s . d . of three independent experiments . ATPase stimulation for GFP - tagged Rad54 , Rdh54NRad54 , Rdh54 or Rad54NRdh54 in the presence of Rad51 - ssDNA . Error bars represent s . d . of three independent experiments . Quantification of Rad51 Atto565 - tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 ( N = 117 ) , Rdh54NRad54 ( N = 150 ) or Rad54NRdh54 ( N = 175 ) . The error bars represent the s . d . for three independent experiments . Fraction translocating Rad51 Atto565 - tailed duplex PSCs in reactions with Rad54 ( N = 117 ) , Rdh54NRad54 ( N = 150 ) , Rad54NRdh54 ( N = 0 ) and Rdh54 ( N = 0 ) . Translocation velocities of Rad51 Atto565 - tailed duplex PSCs in reactions with Rad54 ( N = 86 ) and Rdh54NRad54 ( N = 18 ) . The error bars represent the s . d . of the data .

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

Figure 6D-loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 (300 nM), tailed duplex substrate (15 nM) and a pUC19 plasmid dsDNA (0.3 nM).   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.   D-loop stimulation by Rdh54 and Rad54NRdh54.   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.   ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments.Quantification of Rad51 Atto565-tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 (N = 117), Rdh54NRad54 (N = 150) or Rad54NRdh54 (N=175). The error bars represent the s.d. for three independent experiments.Fraction translocating Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 117), Rdh54NRad54 (N = 150), Rad54NRdh54 (N = 0) and Rdh54 (N = 0).Translocation velocities of Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 86) and Rdh54NRad54 (N = 18). The error bars represent the s.d. of the data.


Figure 7Yeast spot growth assays to monitor the ability of Rad54 , Rdh54 , Rad54NRdh54 , and Rdh54NRad54 to complement MMS sensitivity in rad54 ∆ yeast strains . Yeast spot growth assays to monitor the ability of Rad54 , Rdh54 , Rad54NRdh54 , and Rdh54NRad54 to complement MMS sensitivity in rdh54 ∆ yeast strains .

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Figure 7Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rad54∆ yeast strains.   Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rdh54∆ yeast strains.


Fig 1H & amp ; amp ; E staining of adult head sections of ( A ) repo > GFP . nls and ( B ) repo > Shits1 at 29 ° C for 3 days . Lamina degeneration was identified as vacuoles in ( B ) . Cryosection of adult ( C ) repo > H2B - RFP exhibited the expression of the nuclear red fluorescent protein ( RFP ) in the glia ( epithelialglialnucleus : arrowhead ) and ( D ) repo > Shits1 at 28 ° C for 12 days . Vacuoles were identified in the lamina neuropile ( D ) . Epithelial ( arrowhead ) ( E ) , marginal ( arrow ) and distal satellite ( arrowhead ) glianuclei ( F ) are labeled by HisCl - GAL4 and NP2109 - GAL4 , respectively . Note that HisCl - GAL4 is not expressed in all epithelialglia . ( G , H ) Weak lamina vacuolization was identified in HisCl > Shits1 ( G , 0 . 92 % ) and NP2109 > Shits1 ( H , 1 . 46 % ) at 29 ° C for 14 days . ( I , J ) A single MARCMglia clone ( GFP , green ) expressed Shits1 at 21 ° C ( I ) and 29 ° C ( J ) . ( J ) A vacuole occurred within a glial clone . DAPI ( white ) stains the nuclei ( C , D , and G - J ) . ( K ) The percentage of the vacuole area in the lamina of the repo > Shits1flies at 28 ° C progressively increased . n indicated in each column . P - values were calculated using one - way ANOVA with Bonferroni ' s post - test . ( L ) When repo > Shits1flies were shifted to 28 ° C for 12 days and then shifted to 17 ° C for 9 additional days , the vacuolization was not alleviated . P - values were calculated using one - way ANOVA with Tukey ' s post - test . n indicated in each column .

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

Fig 1H&amp;E staining of adult head sections of (A) repo>GFP.nls and (B) repo>Shits1 at 29°C for 3 days. Lamina degeneration was identified as vacuoles in (B). Cryosection of adult (C) repo>H2B-RFP exhibited the expression of the nuclear red fluorescent protein (RFP) in the glia (epithelialglialnucleus: arrowhead) and (D) repo>Shits1 at 28°C for 12 days. Vacuoles were identified in the lamina neuropile (D). Epithelial (arrowhead) (E), marginal (arrow) and distal satellite (arrowhead) glianuclei (F) are labeled by HisCl-GAL4 and NP2109-GAL4, respectively. Note that HisCl-GAL4 is not expressed in all epithelialglia. (G, H) Weak lamina vacuolization was identified in HisCl>Shits1 (G, 0.92%) and NP2109>Shits1 (H, 1.46%) at 29°C for 14 days. (I, J) A single MARCMglia clone (GFP, green) expressed Shits1 at 21°C (I) and 29°C (J). (J) A vacuole occurred within a glial clone. DAPI (white) stains the nuclei (C, D, and G-J). (K) The percentage of the vacuole area in the lamina of the repo>Shits1flies at 28°C progressively increased. n indicated in each column. P-values were calculated using one-way ANOVA with Bonferroni's post-test. (L) When repo>Shits1flies were shifted to 28°C for 12 days and then shifted to 17°C for 9 additional days, the vacuolization was not alleviated. P-values were calculated using one-way ANOVA with Tukey's post-test. n indicated in each column.


Fig 2 ( A - E ) Horizontal sections of the adult head lamina cartridge . ( A ) In repo > GFP . nls flies , two lamina neurons L1 , L2 axons ( L ) and R1 - 6 axons ( blue area ) were surrounded by the electron - dense cytoplasm of the epithelial glia ( G ) . The section was examined at three different depths , and the size of a single cartridge is not significantly different at different depths . ( B - D ) repo > Shits1 adults maintained at 29 ° C for 4 days . Axons were enlarged but intact . A large number of small and medium vacuoles were identified in the epithelial glia . ( C ) Small vacuoles in the glia that contained a double - membrane structure ( arrowhead ) . ( C ' ) Higher magnification of the boxed area in ( C ) . ( D ) A double - membrane autophagosome - like structure ( arrowhead ) in the glialcytoplasm . ( E ) In repots > DERDN adults maintained at 29 ° C for 2 days , large vacuoles were identified in between the lamina cartridges . N : glialnuclei . ( E ' ) Higher magnification of the boxed area in ( E ) , which shows autophagosome - like vesicles ( arrowhead ) in the gliacytoplasm .

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

Fig 2(A-E) Horizontal sections of the adult head lamina cartridge. (A) In repo>GFP.nls flies, two lamina neurons L1, L2 axons (L) and R1-6 axons (blue area) were surrounded by the electron-dense cytoplasm of the epithelial glia (G). The section was examined at three different depths, and the size of a single cartridge is not significantly different at different depths. (B-D) repo>Shits1 adults maintained at 29°C for 4 days. Axons were enlarged but intact. A large number of small and medium vacuoles were identified in the epithelial glia. (C) Small vacuoles in the glia that contained a double-membrane structure (arrowhead). (C') Higher magnification of the boxed area in (C). (D) A double-membrane autophagosome-like structure (arrowhead) in the glialcytoplasm. (E) In repots>DERDN adults maintained at 29°C for 2 days, large vacuoles were identified in between the lamina cartridges. N: glialnuclei. (E') Higher magnification of the boxed area in (E), which shows autophagosome-like vesicles (arrowhead) in the gliacytoplasm.


Fig 3 ( A ) Rh1 > lacZ + H2B - RFP exhibited nuclearRFP expression in the R1 - 6 photoreceptors ( red ) . ( B ) Rh1 > lacZ + Shits1 maintained at 29 ° C for 14 days resulted in lamina vacuolization . ( C ) Pan - Rh7 > H2B - RFP exhibited expression in R7 ( arrow ) and R8 ( arrowhead ) ( red ) . ( D ) Pan - Rh7 > Shits1 incubated at 29 ° C for 14 days did not exhibit lamina degeneration . ( E ) Lamina monopolar ( L ) neurons ( red ) were selectively labeled by the Ln - GAL4 driven H2B - RFP with repo - GAL80 . ( F ) Ln > Shits1 , repoGAL80 shifted to 29 ° C for 14 days did not exhibit lamina degeneration . ( G ) R1 - 6 photoreceptors were killed in Rh1ts > lacZ + Hid shifted to 29 ° C for 14 days . Rh1ts indicates tubGAL80ts ; Rh1 - GAL4 . Degeneration was induced in the lamina in addition to the retina . ( H ) rdgC306 / + mutant exposed to constant light for 14 days exhibited degeneration in the retina , as well as the lamina . DAPI : nuclei ( white in A - H ) . ( I , K ) Rh1 > lacZ + Shits1 at 29 ° C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus . Arrow : vacuole with internal debris . Arrowhead : large vacuole . ( J ) Lamina cartridge of Rh1 > lacZ + H2B - RFP . N indicates glialnuclei . ( L ) The percentage of the vacuole area in the lamina at 29 ° C for 14 days was examined . All P - values were calculated using one - way ANOVA with Tukey ' s post - test . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 3(A) Rh1>lacZ+H2B-RFP exhibited nuclearRFP expression in the R1-6 photoreceptors (red). (B) Rh1>lacZ+Shits1 maintained at 29°C for 14 days resulted in lamina vacuolization. (C) Pan-Rh7>H2B-RFP exhibited expression in R7 (arrow) and R8 (arrowhead) (red). (D) Pan-Rh7>Shits1 incubated at 29°C for 14 days did not exhibit lamina degeneration. (E) Lamina monopolar (L) neurons (red) were selectively labeled by the Ln-GAL4 driven H2B-RFP with repo-GAL80. (F) Ln>Shits1, repoGAL80 shifted to 29°C for 14 days did not exhibit lamina degeneration. (G) R1-6 photoreceptors were killed in Rh1ts>lacZ+Hid shifted to 29°C for 14 days. Rh1ts indicates tubGAL80ts; Rh1-GAL4. Degeneration was induced in the lamina in addition to the retina. (H) rdgC306/+ mutant exposed to constant light for 14 days exhibited degeneration in the retina, as well as the lamina. DAPI: nuclei (white in A-H). (I, K) Rh1>lacZ+Shits1 at 29°C for 4 days exhibited vacuoles in the electron dense cytoplasm of epithelialglia or near the glianucleus. Arrow: vacuole with internal debris. Arrowhead: large vacuole. (J) Lamina cartridge of Rh1>lacZ+H2B-RFP. N indicates glialnuclei. (L) The percentage of the vacuole area in the lamina at 29°C for 14 days was examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


Fig 4 ( A ) repots > Shits1 + H2B - RFP , ( B ) repots > Shits1 + Egfrλtop and ( C ) repots > Shits1 + RlSem incubated for 12 days at 28 ° C . ( D ) repots > DERDN for 7 days at 28 ° C . ( E ) Epithelial glia MARCM clone and ( F ) Egfrco mutant glia MARCM clone ( labeled with Tomato , green ) . The penetrance is indicated as the number of samples with vacuoles over the total number of samples . DAPI : nuclei ( white in A - F ) . ( G ) Percentage of vacuole area in the lamina neuropile of ( A - D ) . The P - values were calculated using one - way ANOVA with Dunnett ' s post - test . ( H ) Percentage of vacuole area in the lamina of repots > Shits1 flies in the indicated genetic background . The adults were shifted to 28 ° C for 12 days . The P - values were calculated using one - way ANOVA with Tukey ' s post - test . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 4(A) repots>Shits1+H2B-RFP, (B) repots>Shits1+Egfrλtop and (C) repots>Shits1+RlSem incubated for 12 days at 28°C. (D) repots>DERDN for 7 days at 28°C. (E) Epithelial glia MARCM clone and (F) Egfrco mutant glia MARCM clone (labeled with Tomato, green). The penetrance is indicated as the number of samples with vacuoles over the total number of samples. DAPI: nuclei (white in A-F). (G) Percentage of vacuole area in the lamina neuropile of (A-D). The P-values were calculated using one-way ANOVA with Dunnett's post-test. (H) Percentage of vacuole area in the lamina of repots>Shits1 flies in the indicated genetic background. The adults were shifted to 28°C for 12 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. Scale bar: 20 μm.


Fig 5 ( A , A ' ) Anti - Spitz ( red ) immunostaining of w1118 adult head . Spitz can be detected in the retina and lamina . ( B , B ' ) The full - length transmembrane form of Spitz - GFP ( mSpi , green ) expressed in the retina in GMR > mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge . mSpitz - GFP requires processing by Rho and Star to become a secreted form . Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho / Star [ 56 ] ( C ) Knockdown of both EGFR ligands Spi and Krn in R1 - 6 photoreceptors in Rh1 > Dcr2 + Spi - RNAi + Krn - RNAi and ( D ) blockade of Spi processing in Rh1 > lacZ + iRhom exhibited lamina degeneration after shifting to 29 ° C for 14 days . ( E - E ' ) Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28 ° C for 12 days . The clone is labeled by DsRed ( red ) . ( F ) Spi secretion is inhibited in Rh1 > lacZ + Rab11S25N . ( G ) The percentages of the vacuole areas of ( C , D , F ) in lamina at 29 ° C for 14 days were examined . All P - values were calculated using one - way ANOVA with Tukey ' s post - test . DAPI : nuclei ( white in A ' , B ' , C - F ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 5(A, A') Anti-Spitz (red) immunostaining of w1118 adult head. Spitz can be detected in the retina and lamina. (B, B') The full-length transmembrane form of Spitz-GFP (mSpi, green) expressed in the retina in GMR>mSpiGFP flies was predominantly identified in the photoreceptor soma and terminally localized in the lamina cartridge. mSpitz-GFP requires processing by Rho and Star to become a secreted form. Overexpressed mSpitzGFP has been demonstrated to be retained in the perinuclear ER even in the presence of endogenous Rho/Star [56] (C) Knockdown of both EGFR ligands Spi and Krn in R1-6 photoreceptors in Rh1>Dcr2+Spi-RNAi+Krn-RNAi and (D) blockade of Spi processing in Rh1>lacZ+iRhom exhibited lamina degeneration after shifting to 29°C for 14 days. (E-E') Lamina degeneration in whole eye rho7M43ru1 double mutant clones at 28°C for 12 days. The clone is labeled by DsRed (red). (F) Spi secretion is inhibited in Rh1>lacZ+Rab11S25N. (G) The percentages of the vacuole areas of (C, D, F) in lamina at 29°C for 14 days were examined. All P-values were calculated using one-way ANOVA with Tukey's post-test. DAPI: nuclei (white in A', B', C-F). Scale bar: 20 μm.


Fig 6 ( A , A ' ) Anti - Spitz detected Spitz ( magenta ) colocalizing ( arrowhead ) with epithelialgliacytoplasm marked by anti - Black ( green ) . Scale bar : 5 μm . ( B - B ' ) EGFR reporter pointed - lacZ ( pnt - lacZ ) exhibited expression in the epithelial ( arrow ) and marginal glia ( arrowhead ) . ( C , C ' ) Dominant - negative form of EGFR ( DERDN ) expressed in glia at 28 ° C for 3 days inhibited pnt - lacZ expression . ( D , D ' ) pnt - laZ expression was lost in Shits - expressing glia at 28 ° C for 3 days . ( E , E ' ) The knockdown of Spi in R1 - 6 photoreceptors at 28 ° C for 12 days also inhibited pnt - lacZ expression in the glia . The penetrances of ( B - E ) are shown in the upper right corner of each panel . DAPI : nuclei ( white in B ' - E ' ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 6(A, A') Anti-Spitz detected Spitz (magenta) colocalizing (arrowhead) with epithelialgliacytoplasm marked by anti-Black (green). Scale bar: 5 μm. (B-B') EGFR reporter pointed-lacZ (pnt-lacZ) exhibited expression in the epithelial (arrow) and marginal glia (arrowhead). (C, C') Dominant-negative form of EGFR (DERDN) expressed in glia at 28°C for 3 days inhibited pnt-lacZ expression. (D, D') pnt-laZ expression was lost in Shits-expressing glia at 28°C for 3 days. (E, E') The knockdown of Spi in R1-6 photoreceptors at 28°C for 12 days also inhibited pnt-lacZ expression in the glia. The penetrances of (B-E) are shown in the upper right corner of each panel. DAPI: nuclei (white in B'-E'). Scale bar: 20 μm.


Fig 7 ( A - C ) Immunostaining of TUNEL assay ( red ) , active Caspase - 3 ( magenta , A ' - C ' ) and Repo ( green , A " - C " ) . ( A ) GMR - wIR ; repots > lacZ . ( B ) GMR - wIR ; repots > Shits1 . ( C ) GMR - wIR ; repots > DERDN . GMR - wIR was used to reduce the autofluorescence of the eye pigments . Scale bar : 20 μm . ( D - F ) The in vivo fluorescent sensor of caspase activity ( Apoliner ) indicated there was no active caspase activity in the glia . ( D ) repots > Apoliner + lacZ . ( E ) repots > Apoliner + Shits1 . ( F ) repots > Apoliner + DERDN . All adults were shifted to 28 ° C for 5 days . nls - GFP ( green , D - F ) ; merge of GFP ( green ) and mRFP ( red ) in ( D ' - F ' ) ; merge of nls - GFP and DAPI ( magenta ) in ( D " - F " ) . Scale bar : 10 μm . ( G ) Percentage of vacuole area in the lamina in repots > Shits1flies that coexpressed the anti - apoptotic factors P35 and Diap1 . The adults were shifted to 28 ° C for 21 days . The P - values were calculated using one - way ANOVA with Tukey ' s post - test . ( H ) The cell number of the epithelialglia was not reduced in the vacuolated lamina . The numbers of epithelial glia in the lamina of repo > H2B - RFP and repo > H2B - RFP + Shits1 female adults incubated at 28 ° C for 14 days were examined by counting the nuclear RFP at the epithelial layer from the entire Z - stacks of confocal images . P - values were calculated via Mann - Whitney tests .

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

Fig 7(A-C) Immunostaining of TUNEL assay (red), active Caspase-3 (magenta, A'-C') and Repo (green, A"-C"). (A) GMR-wIR; repots>lacZ. (B) GMR-wIR; repots>Shits1. (C) GMR-wIR; repots>DERDN. GMR-wIR was used to reduce the autofluorescence of the eye pigments. Scale bar: 20 μm. (D-F) The in vivo fluorescent sensor of caspase activity (Apoliner) indicated there was no active caspase activity in the glia. (D) repots>Apoliner+lacZ. (E) repots>Apoliner+Shits1. (F) repots>Apoliner+DERDN. All adults were shifted to 28°C for 5 days. nls-GFP (green, D-F); merge of GFP (green) and mRFP (red) in (D'-F'); merge of nls-GFP and DAPI (magenta) in (D"-F"). Scale bar: 10 μm. (G) Percentage of vacuole area in the lamina in repots>Shits1flies that coexpressed the anti-apoptotic factors P35 and Diap1. The adults were shifted to 28°C for 21 days. The P-values were calculated using one-way ANOVA with Tukey's post-test. (H) The cell number of the epithelialglia was not reduced in the vacuolated lamina. The numbers of epithelial glia in the lamina of repo>H2B-RFP and repo>H2B-RFP+Shits1 female adults incubated at 28°C for 14 days were examined by counting the nuclear RFP at the epithelial layer from the entire Z-stacks of confocal images. P-values were calculated via Mann-Whitney tests.


Fig 8In repots > GFP - LC3 + DERDN shifted to 28 ° C , the GFP signal ( green ) was weak on day 1 ( A - A " ) and progressively increased on days 2 ( B - B " ) and 3 ( C - C " ) and colocalized with Ref ( 2 ) P ( magenta ) . ( D ) Ref ( 2 ) P ( magenta ) accumulated within the Egfrco mutant MARCM clone ( green ) . The double - tagged GFP - mCherry - Atg8a is used to distinguish the autophagosomes ( GFP and mCherry , yellow ) and autolysosomes ( mCherry , red ) in autophagic flux . ( E ) In repots > GFP - mCherry - Atg8a + dAtg1 , autophagosomes ( arrowhead ) and autolysosomes ( arrow ) were induced in the glia with normal autophagic flux . ( F ) repots > GFP - mCherry - Atg8a . ( G ) repots > GFP - mCherry - Atg8a + Shits1 . Epithelialglial nuclei are indicated ( arrow ) . ( H ) repots > GFP - mCherry - Atg8a + DERDN . The adults were shifted to 28 ° C for 3 days . GFP : green ( E - H ) ; mCherry : red ( E ' - H ' ) ; merge ( E " - H " ) . Scale bar : 10 μm .

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

Fig 8In repots>GFP-LC3+DERDN shifted to 28°C, the GFP signal (green) was weak on day 1 (A-A") and progressively increased on days 2 (B-B") and 3 (C-C") and colocalized with Ref(2)P (magenta). (D) Ref(2)P (magenta) accumulated within the Egfrco mutant MARCM clone (green). The double-tagged GFP-mCherry-Atg8a is used to distinguish the autophagosomes (GFP and mCherry, yellow) and autolysosomes (mCherry, red) in autophagic flux. (E) In repots>GFP-mCherry-Atg8a+dAtg1, autophagosomes (arrowhead) and autolysosomes (arrow) were induced in the glia with normal autophagic flux. (F) repots>GFP-mCherry-Atg8a. (G) repots>GFP-mCherry-Atg8a+Shits1. Epithelialglial nuclei are indicated (arrow). (H) repots>GFP-mCherry-Atg8a+DERDN. The adults were shifted to 28°C for 3 days. GFP: green (E-H); mCherry: red (E'-H'); merge (E"-H"). Scale bar: 10 μm.


Fig 9 ( A - C ) repots > GFP - LAMP1 . ( D - F ) repots > GFP - LAMP1 + Shits1 . ( G - I ) repots > GFP - LAMP1 + DERDN . Adults were incubated at 28 ° C for 12 h ( A , D , G ) , 24 h ( B , E , H ) , and 48 h ( C , F , I ) , respectively . The GFP - LAMP1 signal ( green ) was induced at 12 h and progressively enhanced in ( D - F ) and ( G - I ) . ( J ) repots > Rab7 - mCherry + H2B - RFP . ( K ) repots > Rab7 - mCherry + Shits1 . ( L ) repots > Rab7 - mCherry + DERDN . The adults were shifted to 28 ° C for 2 days . Rab7 - mCherry puncta ( red ) were increased in ( K , L ) . DAPI : nuclei ( white in J ' - L ' ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 9(A-C) repots>GFP-LAMP1. (D-F) repots>GFP-LAMP1+Shits1. (G-I) repots>GFP-LAMP1+DERDN. Adults were incubated at 28°C for 12 h (A, D, G), 24 h (B, E, H), and 48 h (C, F, I), respectively. The GFP-LAMP1 signal (green) was induced at 12 h and progressively enhanced in (D-F) and (G-I). (J) repots>Rab7-mCherry+H2B-RFP. (K) repots>Rab7-mCherry+Shits1. (L) repots>Rab7-mCherry+DERDN. The adults were shifted to 28°C for 2 days. Rab7-mCherry puncta (red) were increased in (K, L). DAPI: nuclei (white in J'-L'). Scale bar: 20 μm.


Fig 10 ( A ) repots > Rab5S43N . ( B ) repots > Rab7T22N . ( C ) repots > Rab11S25N . ( D - H ) MARCM clones labeled by RFP or GFP ( green ) of control ( D ) , Rab52 ( E ) , α - Ada3 ( F ) , HrsD28 ( G ) , Rab7KO ( H ) , dor8 ( I ) and carΔ146 ( J ) . The penetrance ( number of samples with vacuole over the number of samples examined ) is indicated in each panel . Adults of all genotypes were incubated at 28 ° C for 14 days . DAPI : nuclei ( white in A - J ) . Scale bar : 20 μm . ( K ) The percentage of the vacuole area in ( A - C ) was summarized . Adults of these genotypes were incubated at 28 ° C for 12 days . P - values were calculated using Kruskal - Wallis with Dunn ' s post - tests .

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

Fig 10(A) repots>Rab5S43N. (B) repots>Rab7T22N. (C) repots>Rab11S25N. (D-H) MARCM clones labeled by RFP or GFP (green) of control (D), Rab52 (E), α-Ada3 (F), HrsD28 (G), Rab7KO (H), dor8 (I) and carΔ146 (J). The penetrance (number of samples with vacuole over the number of samples examined) is indicated in each panel. Adults of all genotypes were incubated at 28°C for 14 days. DAPI: nuclei (white in A-J). Scale bar: 20 μm. (K) The percentage of the vacuole area in (A-C) was summarized. Adults of these genotypes were incubated at 28°C for 12 days. P-values were calculated using Kruskal-Wallis with Dunn's post-tests.


Fig 11repots > DERDN was coexpressed with ( A ) H2B - RFP , ( B ) Dorwt , ( C ) Dor - RNAi , ( D ) H2B - RFP , ( E ) Car , ( F ) Car - RNAi , ( G ) lacZ , ( H ) Car - RNAi + Dor - RNAi and ( I ) Car + Dorwt . The autophagy reporter Ref ( 2 ) P ( green ) was stained ( G ' - H ' ) . Scale bar : 20 μm . ( J , K , L ) The percentages of the vacuole areas in the lamina of ( A - C , D - F , G - I ) were summarized , respectively . The adults were shifted to 28 ° C for 7 days . DAPI : nuclei ( white in A - I ) . All P - values were calculated using Kruskal - Wallis tests with Dunn ' s post - tests .

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

Fig 11repots>DERDN was coexpressed with (A) H2B-RFP, (B) Dorwt, (C) Dor-RNAi, (D) H2B-RFP, (E) Car, (F) Car-RNAi, (G) lacZ, (H) Car-RNAi+Dor-RNAi and (I) Car+Dorwt. The autophagy reporter Ref(2)P (green) was stained (G'-H'). Scale bar: 20 μm. (J, K, L) The percentages of the vacuole areas in the lamina of (A-C, D-F, G-I) were summarized, respectively. The adults were shifted to 28°C for 7 days. DAPI: nuclei (white in A-I). All P-values were calculated using Kruskal-Wallis tests with Dunn's post-tests.


Figure 1B , Multiplexed activation of Ascl1 , Lmx1a , NeuroD1 and of Ascl1 , Lmx1a , Nr4a2 in primary astrocytic cultures . n = 3 , representative experiment from 2 - 3 independent experiments , additional data in supplement . Activation levels are depicted as fold change between cells transfected with and without sgRNAs . All levels were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Multiplexed activation of Ascl1, Lmx1a, NeuroD1 and of Ascl1, Lmx1a, Nr4a2 in primary astrocytic cultures. n=3, representative experiment from 2-3 independent experiments, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.