E , F Percentages of CD11b + Ly6G + G - MDSCs gated from total cells in ( E ) spleen and ( F ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 antibody [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . G , H Percentages of CD8 + T cells gated from total cells in ( G ) spleen and ( H ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . I , J Percentages of CD4 + T cells gated from total cells in ( I ) spleen and ( J ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . Flow cytometric analysis of G - MDSCs and T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - IL - 6 treatment after tumor challenge .

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

E, F Percentages of CD11b+ Ly6G+ G-MDSCs gated from total cells in (E) spleen and (F) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 antibody [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. G, H Percentages of CD8+ T cells gated from total cells in (G) spleen and (H) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. I, J Percentages of CD4+ T cells gated from total cells in (I) spleen and (J) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. Flow cytometric analysis of G-MDSCs and T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-IL-6 treatment after tumor challenge.


A . Pull - down experiments using as a bait the biotinylated peptides mimicking the N - terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot ( LxxR motif in red , biotin at the C - terminus ) . Immunoblotting shows affinity - captured GOLPH3 ( endogenous and recombinant ) .

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

A. Pull-down experiments using as a bait the biotinylated peptides mimicking the N-terminal cytosolic tail of glycoenzymes performed from the whole HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail sequence of each glycoenzyme is indicated on the top of the blot (LxxR motif in red, biotin at the C-terminus). Immunoblotting shows affinity-captured GOLPH3 (endogenous and recombinant).


B . Control or GOLPH3 - KD HeLa cells expressing luminal SI - GFP or the chimeras of sucrose - isomaltase containing the N - terminal cytosolic tails of LCS , Gb3S , GM3S and GD3S were fixed and labelled for surface localized GFP ( red ) . Scale bar , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Control or GOLPH3-KD HeLa cells expressing luminal SI-GFP or the chimeras of sucrose-isomaltase containing the N-terminal cytosolic tails of LCS, Gb3S, GM3S and GD3S were fixed and labelled for surface localized GFP (red). Scale bar, 20 μm.


C . Pull - down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates ( top blot ) or purified His - tagged GOLPH3 protein ( bottom blot ) . The N - terminal cytosolic tail of LCS WT and LCS mutants ( mut . 1 , 2 and 3 ) is shown on the top of the blot ( mutations are in blue , LxxR motif in red , biotin is at the C - terminus ) .

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

C. Pull-down experiments of biotinylated LCS WT and LCS mutant cytosolic tails from HeLa cells lysates (top blot) or purified His-tagged GOLPH3 protein (bottom blot). The N-terminal cytosolic tail of LCS WT and LCS mutants (mut. 1, 2 and 3) is shown on the top of the blot (mutations are in blue, LxxR motif in red, biotin is at the C-terminus).


D . HeLa cells expressing luminal SI - GFP chimeras of sucrose - isomaltase ( SI - GFP , green ) containing the WT and mutant N - terminal cytosolic tails of LCS , were fixed and labelled for surface localized GFP ( red ) . Cells expressing LCS - SI - RxR and LCS - SI - LxxR chimeras shown PM staining . Scale bar , 20 μm , ( left ) . PM ( surface ) fluorescence intensity ( in Table S10 are listed the software used in this study ) of each chimera is normalised on the LCS - SI - GFP surface intensity ( data are means ± SEM derived from two biological replicates ; n is indicated in the graph , * * * p < 0 . 001 [ Student ' s t - test ] ) ( right ) .

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

D. HeLa cells expressing luminal SI-GFP chimeras of sucrose-isomaltase (SI-GFP, green) containing the WT and mutant N-terminal cytosolic tails of LCS, were fixed and labelled for surface localized GFP (red). Cells expressing LCS-SI-RxR and LCS-SI-LxxR chimeras shown PM staining. Scale bar, 20 μm, (left). PM (surface) fluorescence intensity (in Table S10 are listed the software used in this study) of each chimera is normalised on the LCS-SI-GFP surface intensity (data are means ± SEM derived from two biological replicates; n is indicated in the graph, ***p <0.001 [Student's t-test]) (right).


E . Quantitative flow cytometry - based analysis of the experiment reported in D ; n is indicated in the graph ( left ) . Percentage of cells with plasma membrane staining ( surface ) is reported for each chimera ( right ) .

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

E. Quantitative flow cytometry-based analysis of the experiment reported in D; n is indicated in the graph (left). Percentage of cells with plasma membrane staining (surface) is reported for each chimera (right).


F . GOLPH3 - KD or OE HeLa cells were processed for ShTxB ( upper panels ) or ChTxB ( lower panels ) staining followed by flow cytometry analysis . Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition .

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

F. GOLPH3-KD or OE HeLa cells were processed for ShTxB (upper panels) or ChTxB (lower panels) staining followed by flow cytometry analysis. Flow cytometry distributions and the relative scatter plots reporting the percentage of positive cells are shown for each condition.


A . HeLa cells mock - treated or GOLPH3 - KD were transiently transfected with LCS _ GFP _ RUSH ( for simplicity , referred in the text as LCS - GR ) and subjected to the synchronisation protocol described in Materials and Methods . Cells were then subjected to IF at each indicated time point : LCS - GR ( green ) , cis Golgi marker GM130 ( red ) , and TGN marker TGN46 ( blue ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . LCS - GR intra - Golgi trafficking in mock or GOLPH3 - KD conditions it has been quantified ( right graph ) by computational coalescence of line scans , and was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) . The n indicated in the graph refers to the number of Golgi stacks analysed in each condition .

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

A. HeLa cells mock-treated or GOLPH3-KD were transiently transfected with LCS_GFP_RUSH (for simplicity, referred in the text as LCS-GR) and subjected to the synchronisation protocol described in Materials and Methods. Cells were then subjected to IF at each indicated time point: LCS-GR (green), cis Golgi marker GM130 (red), and TGN marker TGN46 (blue). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. LCS-GR intra-Golgi trafficking in mock or GOLPH3-KD conditions it has been quantified (right graph) by computational coalescence of line scans, and was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1). The n indicated in the graph refers to the number of Golgi stacks analysed in each condition.


B . Mock and GOLPH3 - KD HeLa cells co - expressing LCS - GR and VSV - G were subjected to the co - synchronisation protocol described in Materials and Methods . Cells were then subjected to IF : LCS - GR or VSV - G ( green ) , GM130 ( blue ) , and TGN46 ( red ) . Representative individual Golgi mini - stacks are shown . Scale bar , 1 µm . C . Quantification of the experiment in ( B ) was performed by normalized line scan analysis ( normalisation of the distances was performed considering the GM130 peak as 0 , and the TGN46 peak as 1 ) ; n indicated in the graph refers to the number of Golgi stacks analysed in each condition , individual data points and means ± SEM are shown , * * * p < 0 . 001 ( Student ' s t test ) ; ns , not significant . D

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mock and GOLPH3-KD HeLa cells co-expressing LCS-GR and VSV-G were subjected to the co-synchronisation protocol described in Materials and Methods. Cells were then subjected to IF: LCS-GR or VSV-G (green), GM130 (blue), and TGN46 (red). Representative individual Golgi mini-stacks are shown. Scale bar, 1 µm. C. Quantification of the experiment in (B) was performed by normalized line scan analysis (normalisation of the distances was performed considering the GM130 peak as 0, and the TGN46 peak as 1); n indicated in the graph refers to the number of Golgi stacks analysed in each condition, individual data points and means ± SEM are shown, ***p < 0.001 (Student's t test); ns, not significant. D


D . HeLa cells were transfected with LCS - GR , subjected to the synchronisation protocol ( see Materials and Methods ) , fixed and processed for cryoimmunolabeling with an anti - GFP antibody ( 15 nm gold particles ) and anti - GM130 antibody ( 10 nm gold particles , black arrowheads ) peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . Scale bar , 150 nm .

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

D. HeLa cells were transfected with LCS-GR, subjected to the synchronisation protocol (see Materials and Methods), fixed and processed for cryoimmunolabeling with an anti-GFP antibody (15 nm gold particles) and anti-GM130 antibody (10 nm gold particles, black arrowheads) peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. Scale bar, 150 nm.


E , F . Quantification of the distribution of LCS - GR as measured by Frequency Distribution Analysis ( E ) and linear density ( LD , in F ) ( data are means ± SEM , number of Golgi stacks is n = 15 at 0 min , n = 14 at 10 min , n = 17 at 20 min . ( E ) The red arrows indicate the fraction of LCS - GR gold particles ( % ) in vesicles at each indicated time - point .

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

E, F. Quantification of the distribution of LCS-GR as measured by Frequency Distribution Analysis (E) and linear density (LD, in F) (data are means± SEM, number of Golgi stacks is n=15 at 0 min, n=14 at 10 min, n=17 at 20 min. (E) The red arrows indicate the fraction of LCS-GR gold particles (%) in vesicles at each indicated time-point.


G . HeLa cells expressing LCS - GR mock or GOLPH3 - KD were treated and stained as in D . Black arrowheads indicate GM130 ; peri - Golgi vesicles containing LCS - GR are marked by red arrowheads . LCS - GR in the TGN is marked by blue arrowheads . Scale bar , 150 nm .

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

G. HeLa cells expressing LCS-GR mock or GOLPH3-KD were treated and stained as in D. Black arrowheads indicate GM130; peri-Golgi vesicles containing LCS-GR are marked by red arrowheads. LCS-GR in the TGN is marked by blue arrowheads. Scale bar, 150 nm.


H . Quantification of LCS - GR distribution ( linear density , LD ) across the stack and in peri - Golgi vesicles ( data are means ± SEM , number of Golgi stacks is n = 17 at 20 min for the CTRL , n = 13 at 20 min for the GOLPH3 KD ; * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

H. Quantification of LCS-GR distribution (linear density, LD) across the stack and in peri-Golgi vesicles (data are means ± SEM, number of Golgi stacks is n=17 at 20 min for the CTRL, n=13 at 20 min for the GOLPH3 KD; *p <0.05, **p < 0.01; ***p <0.001 [Student's t-test]).


A . PHFs GOLPH3 OE ( left ) , or KD ( right ) , were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , TGN46 or DAPI ( blue ) . Dashed lines indicate cells overexpressing or interfered for GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ; data are means ± SEM derived from two biological replicates , n = 42 in CTRL , n = 49 in GOLPH3 KD and n = 27 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

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

A. PHFs GOLPH3 OE (left), or KD (right), were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), TGN46 or DAPI (blue). Dashed lines indicate cells overexpressing or interfered for GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi; data are means ± SEM derived from two biological replicates, n=42 in CTRL, n=49 in GOLPH3 KD and n=27 in GOLPH3 OE; ***p <0.001 [Student's t-test]).


B . GOLPH3 - OE HeLa cells were fixed and processed for IF : Endogenous LCS ( green ) , GOLPH3 ( red ) , and TGN46 ( blue ) . Dashed lines indicate cells overexpressing GOLPH3 . Scale bar , 20 µm . Quantification of the amount of endogenous LCS ( right graph ) at the Golgi ( data are means ± SEM derived from two biological replicates , n = 21 in CTRL , n = 11 in GOLPH3 OE ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GOLPH3-OE HeLa cells were fixed and processed for IF: Endogenous LCS (green), GOLPH3 (red), and TGN46 (blue). Dashed lines indicate cells overexpressing GOLPH3. Scale bar, 20 µm. Quantification of the amount of endogenous LCS (right graph) at the Golgi (data are means ± SEM derived from two biological replicates, n=21 in CTRL, n=11 in GOLPH3 OE; ***p <0.001 [Student's t-test]).


C . GOLPH3 OE or KD HeLa cells expressing LCS - HA were fixed and processed for IF : HA ( green ) . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm .

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

C. GOLPH3 OE or KD HeLa cells expressing LCS-HA were fixed and processed for IF: HA (green). Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.


D . Assessment of LCS - GR synchronized transport in GOLPH3 OE or KD HeLa cells . Micrographs show cells at the indicated trafficking time points . Scale bar , 20 µm .

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

D. Assessment of LCS-GR synchronized transport in GOLPH3 OE or KD HeLa cells. Micrographs show cells at the indicated trafficking time points. Scale bar, 20 µm.


E . Representative WB of cells treated as in ( D ) ( n = 3 ) .

E. Representative WB of cells treated as in (D) (n = 3).


F . Plots of the densitometric quantification of the amount of LCS - GR ( Golgi mature band ) over time , starting from 2 hr chase ( data are means of at least 3 experiments ± SD ) .

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

F. Plots of the densitometric quantification of the amount of LCS-GR (Golgi mature band) over time, starting from 2 hr chase (data are means of at least 3 experiments ± SD).


G . GOLPH3 OE or KD HeLa cells expressing LCS - HA were treated with bafilomycin A1 ( BafA1 , 10 nM ) for 16 hr , fixed , and processed for IF labelling : LCS ( green ) , LAMP1 ( red ) , and DAPI ( blue ) . Arrowheads indicate LCS and LAMP1 co - localisation . Scale bar , 20 µm .

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

G. GOLPH3 OE or KD HeLa cells expressing LCS-HA were treated with bafilomycin A1 (BafA1, 10 nM) for 16 hr, fixed, and processed for IF labelling: LCS (green), LAMP1 (red), and DAPI (blue). Arrowheads indicate LCS and LAMP1 co-localisation. Scale bar, 20 µm.


H . WB of cells treated as in ( G ) . Densitometric quantification of the blot is plotted in the top panel ( data are means of at least 3 experiments ± SD ) .

H. WB of cells treated as in (G). Densitometric quantification of the blot is plotted in the top panel (data are means of at least 3 experiments ± SD).


I . GOLPH3 OE or KD HeLa cells expressing the indicated HA - tagged glycoenzymes were fixed and processed for IF . Asterisks indicate GOLPH3 OE cells . Scale bar , 20 µm .

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

I. GOLPH3 OE or KD HeLa cells expressing the indicated HA-tagged glycoenzymes were fixed and processed for IF. Asterisks indicate GOLPH3 OE cells. Scale bar, 20 µm.


J . Number of glycoenzymes - HA positive spots per cell in GOLPH3 - KD cells ( left panel ) and percentage of cells with glycoenzymes - HA in the ER in GOLPH3 - OE cells ( right panel ) are plotted ( n is indicated in the graph ; data are means ± SEM ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

J. Number of glycoenzymes-HA positive spots per cell in GOLPH3-KD cells (left panel) and percentage of cells with glycoenzymes-HA in the ER in GOLPH3-OE cells (right panel) are plotted (n is indicated in the graph; data are means ± SEM; ***p <0.001 [Student's t-test]).


A . Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [ 3H ] - sphingosine pulse ( 2 hr ) and chase ( indicated times ) , lipid extraction and HPTLC separation and radioactive counting ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) .

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

A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]).


B . Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on sphingolipid levels as assessed by LC / MS of HeLa cells ( bottom ) ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


C . Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI - MS after lipid extraction . Profiles are representative of 3 independent experiments . Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID : ST001674 .

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

C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674.


D . Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig . 1F ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 [ Student ' s t - test ] ) .

D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]).


E . Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( top ) and PHFs ( middle ) . Effects of LCS KD or OE on ceramide levels as assessed by LC / MS of HeLa cells ( bottom ) . The dotted black box highlights the effects on C16 : 0 Cer ( data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ Student ' s t - test ] ) . Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs : ST001673 for HeLa cells , and ST001672 for PHFs .

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

E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.


A . Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS - PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway . Data are representative of at least 3 independent experiments .

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A. Cell lysates from HeLa cells KD or OE for either GOLPH3 or LCS were processed for SDS-PAGE and Western blotting with antibodies aimed at evaluating the activation status of the mTOR pathway. Data are representative of at least 3 independent experiments.


B . Clonogenic assay of HeLa cells in adherence . 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days . Afterward , colonies were fixed and stained as detailed in Materials and Methods . The graph on right reports the number of colonies per well in each indicated condition ( data are means ± SEM of 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 [ Student ' s t - test ] ) .

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B. Clonogenic assay of HeLa cells in adherence. 250 HeLa cells KD for either GOLPH3 or LCS were allowed to form colonies for 10 days. Afterward, colonies were fixed and stained as detailed in Materials and Methods. The graph on right reports the number of colonies per well in each indicated condition (data are means ± SEM of 3 independent experiments; *p <0.05, **p <0.01 [Student's t-test]).


C . Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 ( left upper graph ) or LCS ( left bottom graph ) encoding plasmids . Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS ( right upper graph ) . Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD ( right bottom graph ) . Colonies were stained and quantified as described in Materials and Methods ; ( individual data points are shown in the graphs ; data are means ± SD of at least 3 independent experiments ; * p < 0 . 05 , * * * p < 0 . 001 , n . s . = not significant [ Student ' s t - test ] ) .

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C. Soft agar colony formation assay of HeLa cells transfected with different concentration of either GOLPH3 (left upper graph) or LCS (left bottom graph) encoding plasmids. Soft agar colony formation assay of HeLa cells KD for either GOLPH3 or LCS (right upper graph). Soft agar colony formation assay of HeLa cells KD for LCS and GOLPH3 OE alone or in combination with LCS KD (right bottom graph). Colonies were stained and quantified as described in Materials and Methods; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001, n.s. = not significant [Student's t-test]).


D . Protein lysates from parental ( left ) , GCS - KO ( middle ) , and LCS - KO ( right ) HeLa cells , CTRL or GOLPH3 - OE were processed for SDS - PAGE and WB with the indicated antibodies . Data are representative of at least 3 independent experiments .

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D. Protein lysates from parental (left), GCS-KO (middle), and LCS-KO (right) HeLa cells, CTRL or GOLPH3-OE were processed for SDS-PAGE and WB with the indicated antibodies. Data are representative of at least 3 independent experiments.


E . Soft agar colony formation assay of parental and LCS - KO HeLa cells expressing LCS - HA or GOLPH3 . Colony number was quantified ; ( individual data points are shown in the graphs ; data are means ± SD of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

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E. Soft agar colony formation assay of parental and LCS-KO HeLa cells expressing LCS-HA or GOLPH3. Colony number was quantified; (individual data points are shown in the graphs; data are means ± SD of at least 3 independent experiments; ***p <0.001 [Student's t-test]).


F . Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS ( data are means ± SEM of at least 3 independent experiments ; * * * p < 0 . 001 [ Student ' s t - test ] ) .

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F. Quantification of soft agar colony formation assay of LNCaP and DU145 prostatic cancer cell lines treated with increasing concentrations of siRNA targeting GOLPH3 or LCS (data are means ± SEM of at least 3 independent experiments; ***p <0.001 [Student's t-test]).


D . Representative images of NSCLC sections stained for GOLPH3 and LCS . Scale bar , 100 µm .

D. Representative images of NSCLC sections stained for GOLPH3 and LCS. Scale bar, 100 µm.


( B ) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm .

(B) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( C ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( B ) , with DAPI staining ( blue ) depicted in addition to PaprikaRFP ( Cas9 ) and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

(C) Higher magnification of the VZ and SVZ of the electroporated area shown in (B), with DAPI staining (blue) depicted in addition to PaprikaRFP (Cas9) and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( D ) Quantification of the proportion of Cas9 - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid electroporation . Data are the mean of 4 independent experiments ( 7 embryos per condition in total , from 4 litters ) .

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(D) Quantification of the proportion of Cas9-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 plasmid electroporation. Data are the mean of 4 independent experiments (7 embryos per condition in total, from 4 litters).


( F ) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence ( magenta ) showing the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm .

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(F) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence (magenta) showing the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( G ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( F ) , with DAPI staining ( blue ) depicted in addition to mCherry and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

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(G) Higher magnification of the VZ and SVZ of the electroporated area shown in (F), with DAPI staining (blue) depicted in addition to mCherry and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


H ) Quantification of the proportion of mCherry - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 protein electroporation . Data are the mean of 4 independent experiments ( 5 embryos per condition in total , from 4 litters ) .

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

H) Quantification of the proportion of mCherry-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 protein electroporation. Data are the mean of 4 independent experiments (5 embryos per condition in total, from 4 litters).


( I ) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

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(I) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( J ) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence ( magenta ) and the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

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(J) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence (magenta) and the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


K ) Quantification of the proportion of Cas9 - positive cells ( left ) and mCherry - positive cells ( right ) in the VZ plus SVZ that are GFP - positive 24 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid ( left ) or protein ( right ) electroporation . Data are the mean of 3 independent experiments each ( 3 embryos per condition in total , from 3 litters ) . ( D , H , K ) Controls were set to 100 % and the gGFP conditions expressed relative to control ( D , 11 % ; H , 24 % ; K left , 40 % K right , 28 % ) . Error bars indicate SD ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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K) Quantification of the proportion of Cas9-positive cells (left) and mCherry-positive cells (right) in the VZ plus SVZ that are GFP-positive 24 h after control (Con, white) or gGFP (black) Cas9 plasmid (left) or protein (right) electroporation. Data are the mean of 3 independent experiments each (3 embryos per condition in total, from 3 litters).(D, H, K) Controls were set to 100% and the gGFP conditions expressed relative to control (D, 11%; H, 24%; K left, 40% K right, 28%). Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.


( B ) Daughter cells of single aRGCs microinjected with either Cas9 / control gRNA ( top ) or Cas9 / gGFP ( bottom ) revealed by Dx - A555immunofluorescence ( magenta ) ; Cells 1 , aRGC daughter ; Cells 2 , BP daughter . Dashed lines indicate ventricular surface . Images are maximum intensity projections of 20 ( Control ) and 24 ( gGFP ) optical sections . Scale bars , 20 µm .

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(B) Daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555immunofluorescence (magenta); Cells 1, aRGC daughter; Cells 2, BP daughter. Dashed lines indicate ventricular surface. Images are maximum intensity projections of 20 (Control) and 24 (gGFP) optical sections. Scale bars, 20 µm.


( C ) Single optical sections of Cells 1 and Cells 2 depicted in ( B ) , showing the effects of Cas9 and control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( as revealed by immunofluorescence , green ) . Cells 1 and Cells 2 are indicated by dotted lines ; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9 / gGFP microinjection . Scale bars , 5 µm .

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(C) Single optical sections of Cells 1 and Cells 2 depicted in (B), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression (as revealed by immunofluorescence, green). Cells 1 and Cells 2 are indicated by dotted lines; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9/gGFP microinjection. Scale bars, 5 µm.


( D ) Quantification of the proportion of daughter cells ( Dx - A555 + ) of microinjected cells that show GFP expression 24 h after control ( Con , white ) or gGFP ( black ) microinjection . Control is set to 100 % and the gGFP condition expressed relative to control ( 39 % ) . Data are the mean of two independent experiments ( 3 embryos and 1 litter per experiment , total number of daughter cells scored : control 142 , gGFP 88 ) ; bars indicate the variation of the two individual values from the mean ( P < 0 . 05 ) .

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(D) Quantification of the proportion of daughter cells (Dx-A555+) of microinjected cells that show GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. Control is set to 100% and the gGFP condition expressed relative to control (39%). Data are the mean of two independent experiments (3 embryos and 1 litter per experiment, total number of daughter cells scored: control 142, gGFP 88); bars indicate the variation of the two individual values from the mean (P<0.05).


( A - D ) Neocortex of mouseE13 . 5embryos was in utero electroporated with a plasmid encoding , under constitutive promoters , Cas9 _ T2A _ PaprikaRFP and gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( Tbr2 ) , followed by analysis at E15 . 5 . ( A ) Electroporated area showing the effects of Cas9 expression ( revealed by PaprikaRFPDAPI , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on aRGCs expression ( green , immunofluorescence ) ; blue , Cas9staining . Boxes indicate areas shown at higher magnification in the insets ( 45 x 45 μm ) . Dotted lines indicate nuclei of progeny of electroporated Cas9 ; note the presence of Tbr2 immunoreactivity in the control ( top ) and its absence upon Cas9 / gTbr2 plasmid electroporation ( bottom ) . Scale bars , 20 μm .

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(A-D) Neocortex of mouseE13.5embryos was in utero electroporated with a plasmid encoding, under constitutive promoters, Cas9_T2A_PaprikaRFP and gRNA targeting either LacZ (Control, Con) or Tbr2 (Tbr2), followed by analysis at E15.5.(A) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFPDAPI, magenta) together with either control gRNA (top) or gTbr2 (bottom) on aRGCs expression (green, immunofluorescence); blue, Cas9staining. Boxes indicate areas shown at higher magnification in the insets (45 x 45 μm). Dotted lines indicate nuclei of progeny of electroporated Cas9; note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 plasmid electroporation (bottom). Scale bars, 20 μm.


( B ) Quantification of the proportion of Cas9 + cells that are Tbr2 + 48 h after control ( Con , white ) or gTbr2 ( black ) plasmid electroporation . Data are the mean of 4 independent experiments ( 6 embryos per condition , from 4 litters ) .

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(B) Quantification of the proportion of Cas9+ cells that are Tbr2+ 48 h after control (Con, white) or gTbr2 (black) plasmid electroporation. Data are the mean of 4 independent experiments (6 embryos per condition, from 4 litters).


( C ) Electroporated area showing the effects of Cas9 expression ( revealed by PaprikaRFP fluorescence , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on the abundance of mitotic progenitors as revealed by phosphohistone H3 ( PH3 ) immunofluorescence ( green ) . Apical and basal mitoses are indicated by arrows and arrowheads , respectively ; note the reduction in mitotic Cas9 + BPs upon Cas9 / gTbr2 plasmid electroporation . Scale bars , 20 μm .

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(C) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFP fluorescence, magenta) together with either control gRNA (top) or gTbr2 (bottom) on the abundance of mitotic progenitors as revealed by phosphohistone H3 (PH3) immunofluorescence (green). Apical and basal mitoses are indicated by arrows and arrowheads, respectively; note the reduction in mitotic Cas9+ BPs upon Cas9/gTbr2 plasmid electroporation. Scale bars, 20 μm.


( D ) Quantification of apical and basal Cas9 + mitoses ( as revealed by PH3 immunofluorescence ) in a unit area 48 h after Cas9 / gTbr2 plasmid electroporation . Data are expressed as percentage of the data obtained upon control plasmid electroporation and are the mean of 3 independent experiments ( 3 embryos per condition in total , from 3 litters ) ; note the reduction in basal ( black , 68 % ) but not apical ( white ) mitoses .

(D) Quantification of apical and basal Cas9+ mitoses (as revealed by PH3 immunofluorescence) in a unit area 48 h after Cas9/gTbr2 plasmid electroporation. Data are expressed as percentage of the data obtained upon control plasmid electroporation and are the mean of 3 independent experiments (3 embryos per condition in total, from 3 litters); note the reduction in basal (black, 68%) but not apical (white) mitoses.


( E - H ) Single aRGCs in neocortex in organotypic slices of telencephalon of E14 . 5 mouse embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( gTbr2 ) and with Dextran 10 , 000 - Alexa 555 ( Dx - A555 ) , followed by analysis after 48 h of culture . ( E ) Progeny of single microinjected aRGCs ( revealed by Dx - A555immunofluorescence , magenta , dotted lines ) showing the effects of Cas9 together with either control gRNA ( top ) or gTbr2 ( bottom ) on Tbr2 expression ( green , immunofluorescence ) ; blue , DAPI staining . Note the presence of Tbr2 immunoreactivity in the control ( top ) and its absence upon Cas9 / gTbr2 ( bottom ) protein microinjection . Images are single optical sections . Scale bars , 5 µm .

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(E-H) Single aRGCs in neocortex in organotypic slices of telencephalon of E14.5 mouse embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ (Control, Con) or Tbr2 (gTbr2) and with Dextran 10,000-Alexa 555 (Dx-A555), followed by analysis after 48 h of culture.(E) Progeny of single microinjected aRGCs (revealed by Dx-A555immunofluorescence, magenta, dotted lines) showing the effects of Cas9 together with either control gRNA (top) or gTbr2 (bottom) on Tbr2 expression (green, immunofluorescence); blue, DAPI staining. Note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 (bottom) protein microinjection. Images are single optical sections. Scale bars, 5 µm.


( F ) Quantification of the proportion of the progeny ( Dx - A555 + ) of microinjected aRGCs that show Tbr2 expression 48 h after control ( Con , white ) or gTbr2 ( black ) microinjection . Data are the mean of 3 independent experiments ( 3 embryos per condition ; total number of cells scored : control 45 , gTbr2 41 ) .

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(F) Quantification of the proportion of the progeny (Dx-A555+) of microinjected aRGCs that show Tbr2 expression 48 h after control (Con, white) or gTbr2 (black) microinjection. Data are the mean of 3 independent experiments (3 embryos per condition; total number of cells scored: control 45, gTbr2 41).


( G ) Distribution across the cortical wall of the progeny ( revealed by Dx - A555immunofluorescence , magenta ; arrows , aRGC ; arrowheads , BPs ; open arrow , neuron ) of single control ( left ) or gTbr2 ( right ) microinjected aRGCs . Dashed lines indicate ventricular surface ( bottom ) and basal lamina ( top ) . Images are maximum intensity projections of 67 ( Control ) and 94 ( gTbr2 ) single optical sections . Scale bars , 20 µm .

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(G) Distribution across the cortical wall of the progeny (revealed by Dx-A555immunofluorescence, magenta; arrows, aRGC; arrowheads, BPs; open arrow, neuron) of single control (left) or gTbr2 (right) microinjected aRGCs. Dashed lines indicate ventricular surface (bottom) and basal lamina (top). Images are maximum intensity projections of 67 (Control) and 94 (gTbr2) single optical sections. Scale bars, 20 µm.


( H ) Quantification of the distribution of the progeny ( Dx - A555 + ) of microinjected aRGCs in the VZ , SVZ and intermediate zone / cortical plate ( CP + IZ ) 48 h after control ( Con , white ) or gTbr2 ( black ) microinjection ( total number of cells scored : control 37 , gTbr2 51 , pooled from at least 3 independent experiments ) . Note that upon gTbr2 microinjection the distribution of cells across the various cortical zones is significantly different ( VZ : control 77 % , gTbr2 39 : SVZ : control 20 % , gTbr2 37 % ; IZ / CP : control 3 % , gTbr2 24 % , * , P < 0 . 05 ) . ( B , F ) Controls were set to 100 % and the gTbr2 conditions expressed relative to control ( B , 48 % ; F , 50 % ) . ( B , D , F ) Error bars indicate SD ( B , D , F ) ; * , P < 0 . 05 ; * * , P < 0 . 01 .

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

(H) Quantification of the distribution of the progeny (Dx-A555+) of microinjected aRGCs in the VZ, SVZ and intermediate zone/cortical plate (CP+IZ) 48 h after control (Con, white) or gTbr2 (black) microinjection (total number of cells scored: control 37, gTbr2 51, pooled from at least 3 independent experiments). Note that upon gTbr2 microinjection the distribution of cells across the various cortical zones is significantly different (VZ: control 77%, gTbr2 39: SVZ: control 20%, gTbr2 37%; IZ/CP: control 3%, gTbr2 24%, *, P<0.05).(B, F) Controls were set to 100% and the gTbr2 conditions expressed relative to control (B, 48%; F, 50%).(B, D, F) Error bars indicate SD (B, D, F); *, P<0.05; **, P<0.01.


Figure 4 . Sequencing analysis of the CRISPR / Cas9 - induced disruption of the Tbr2 locus . ( A - C ) Next - generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off - target loci generated from PaprikaRFP - expressing cells isolated at E15 . 5 from neocortex of mouse embryos in utero electroporated at E13 . 5 with plasmids encoding Cas9 _ T2A _ PaprikaRFP and either gTbr2 ( black ) or control gRNA ( Con , white ) as in Fig 3 A - D . ( A ) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off - target sites ( OT1 - 4 ) . Data are expressed as percentage of the total number of reads and are the mean of two independent experiments ( 8 and 5 embryos per condition , respectively ) . ( B , C ) Analysis of one of the experiments of ( A ) for deletions ( B ) and insertions ( C ) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9 / gTbr2 plasmid electroporation .

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Figure 4. Sequencing analysis of the CRISPR/Cas9-induced disruption of the Tbr2 locus.(A-C) Next-generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off-target loci generated from PaprikaRFP-expressing cells isolated at E15.5 from neocortex of mouse embryos in utero electroporated at E13.5 with plasmids encoding Cas9_T2A_PaprikaRFP and either gTbr2 (black) or control gRNA (Con, white) as in Fig 3 A-D.(A) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off-target sites (OT1-4). Data are expressed as percentage of the total number of reads and are the mean of two independent experiments (8 and 5 embryos per condition, respectively).(B, C) Analysis of one of the experiments of (A) for deletions (B) and insertions (C) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9/gTbr2 plasmid electroporation.


A Levels of ULK1 in MEFs exposed to hypoxia ( 1 % O2 ) for the indicated times . actin , Western

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A Levels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.actin, Western blot


B Quantification of the ratio of ULK1 to actin in ( A ) .

B Quantification of the ratio of ULK1 to actin in (A).


C Levels of ULK1 in MEFs treated with FCCP ( 20 μM ) for the indicated times .

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C Levels of ULK1 in MEFs treated with FCCP (20 μM) for the indicated times.actin


D Quantification of the ratio of ULK1 to actin in ( C ) .

D Quantification of the ratio of ULK1 to actin in (C).


E MEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 6 h and then fixed by 4 % paraformaldehyde . Cells were stained with anti ‐ TIM23 ( mouse ) and anti ‐ ULK1 ( rabbit ) primary antibodies , then Alexa Fluor ‐ 488 ‐ labeled donkey anti ‐ rabbit IgG and Alexa Fluor ‐ 555 ‐ labeled donkey anti ‐ mouse IgG secondary antibodies before analysis by immunofluorescence microscopy . Scale bar , 10 μm .

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E MEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 6 h and then fixed by 4% paraformaldehyde. Cells were stained with anti‐TIM23 (mouse) and anti‐ULK1 (rabbit) primary antibodies, then Alexa Fluor‐488‐labeled donkey anti‐rabbit IgG and Alexa Fluor‐555‐labeled donkey anti‐mouse IgG secondary antibodies before analysis by immunofluorescence microscopy. Scale bar, 10 μm.


F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia ( 1 % O2 ) for 12 h . PNS , post ‐ nuclear supernatant ; Cyto , cytosol ; Mito , mitochondria .

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F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia (1% O2) for 12 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1


G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP ( 20 μM ) for 6 h . PNS , post ‐ nuclear supernatant ; Cyto , cytosol ; Mito , mitochondria .

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G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP (20 μM) for 6 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1


A HeLa cells were co − transfected with FUNDC1 − Myc and FLAG − ULK1 . Cells were lysed and immunoprecipitated with anti − Myc antibody 24 h after transfection .

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A HeLa cells were co−transfected with FUNDC1−Myc and FLAG−ULK1. Cells were lysed and immunoprecipitated with anti−Myc antibody 24 h after transfection.


B FLAG − FUNDC1 was co − transfected with GFP − vector or GFP − ULK1 for 24 h in HeLa cells , and then , cell lysates were immunoprecipitated with anti − GFP antibody .

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B FLAG−FUNDC1 was co−transfected with GFP−vector or GFP−ULK1 for 24 h in HeLa cells, and then, cell lysates were immunoprecipitated with anti−GFP antibody.


CMEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 3 h , followed by immunoprecipitation using anti ‐ ULK1 antibody .

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CMEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 3 h, followed by immunoprecipitation using anti‐ULK1 antibody.


D The expression of FUNDC1 is induced by 10 ng / ml tetracycline ( Tet ) in FUNDC1 ‐ inducible HeLa cells . Cells were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) for 24 h . Then , the cell lysates were prepared for immunoblotting using indicated antibodies .

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D The expression of FUNDC1 is induced by 10 ng/ml tetracycline (Tet) in FUNDC1‐inducible HeLa cells. Cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N) for 24 h. Then, the cell lysates were prepared for immunoblotting using indicated antibodies.


EULK1 + / + cells were transfected with FUNDC1 ‐ Myc , and ULK1 - / - cells were transfected with FUNDC1 ‐ Myc alone , FUNDC1 ‐ Myc and HA ‐ ULK1 , or FUNDC1 ‐ Myc and HA ‐ ULK1 ( K46N ) . Cells were harvested and lysed for immunoblots24 h after transfection .

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EULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1-/- cells were transfected with FUNDC1‐Myc alone, FUNDC1‐Myc and HA‐ULK1, or FUNDC1‐Myc and HA‐ULK1 (K46N). Cells were harvested and lysed for immunoblots24 h after transfection.


F Purified GST ‐ FUNDC1 and GST ‐ FUNDC1 ( S17A ) were subjected to an in vitro kinase assay with Myc ‐ ULK1 immunoprecipitated by anti ‐ Myc antibody from Myc ‐ ULK1 ‐ transfected cells about 24 h post ‐ transfection . Phosphorylated FUNDC1 was detected by anti ‐ p ‐ S17 ‐ FUNDC1 antibody . The red asterisks mark the target bands .

F Purified GST‐FUNDC1 and GST‐FUNDC1 (S17A) were subjected to an in vitro kinase assay with Myc‐ULK1 immunoprecipitated by anti‐Myc antibody from Myc‐ULK1‐transfected cells about 24 h post‐transfection. Phosphorylated FUNDC1 was detected by anti‐p‐S17‐FUNDC1 antibody. The red asterisks mark the target bands.


G Western blotULK1 , FUNDC1 , and the phosphorylated FUNDC1 in MEFs exposed to hypoxia ( 1 % O2 ) for the indicated times .

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G Western blotULK1, FUNDC1, and the phosphorylated FUNDC1 in MEFs exposed to hypoxia (1% O2) for the indicated times.


HWestern blot analysis of the kinetics of ULK1 , FUNDC1 , and the phosphorylated in MEFs treated with FCCP ( 20 μM ) for the indicated times .

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HWestern blot analysis of the kinetics of ULK1, FUNDC1, and the phosphorylated in MEFs treated with FCCP(20 μM) for the indicated times.actin


I , J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified . HeLa cells were co ‐ transfected with Flag ‐ ULK1 and the indicated constructs . 24 h after transfection , cells were lysed for immunoprecipitation by anti ‐ Flag antibody .

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I, J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified. HeLa cells were co‐transfected with Flag‐ULK1 and the indicated constructs. 24 h after transfection, cells were lysed for immunoprecipitation by anti‐Flag antibody.


KMEFs were transfected with FUNDC1 ‐ Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA . 36 h after transfection , cells were harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) and then lysed for immunoblotting with the indicated antibodies .

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KMEFs were transfected with FUNDC1‐Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA. 36 h after transfection, cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1) and then lysed for immunoblotting with the indicated antibodies.


L MEFs were co ‐ transfected with FUNDC1 ‐ Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA . 24 h post ‐ transfection , cells were treated with hypoxic ( 1 % O2 ) conditions for 12 h or FCCP ( 20 μM ) for 6 h before fixed by 4 % paraformaldehyde and stained with indicated antibodies . Scale bar , 10 μm .

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L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.


AULK1 + / + and ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


B ULK1 + / + and ULK1 − / − cells were treated with FCCP ( 20 μM ) for the indicated times in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


C ULK1 + / + and ULK1 − / − cells were transfected with FUNDC1 − Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


D ULK1 − / − cells were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells lysates were immunoblotted .

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D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted.


EULK1 − / − cells were transfected with HA − ULK1 or HA − ULK1 and then treated with FCCP ( 20 μM ) for 24 h . Cells were harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


F FUNDC1 ‐ knockdown MEFs were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17A ) for the indicated times . Cell lysates were used for immunoblotting .

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

F FUNDC1‐knockdown MEFs were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17A) for the indicated times. Cell lysates were used for immunoblotting.


G ULK1 + / + cells were transfected with FUNDC1 ‐ Myc , and ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cells were fixed with 4 % paraformaldehyde and stained with anti ‐ Myc ( mouse ) and anti ‐ LC3 ( rabbit ) primary antibodies for immunofluorescence microscopy . Scale bar , 10 μm .

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G ULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D). 24 h after transfection, cells were fixed with 4% paraformaldehyde and stained with anti‐Myc (mouse) and anti‐LC3 (rabbit) primary antibodies for immunofluorescence microscopy. Scale bar, 10 μm.


H ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were immunoblotted .

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H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted.


I ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were detected with the citrate synthase activity kit .

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I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit.


J HeLa cells were transfected with FUNDC1 ‐ Myc , FUNDC1 - Myc ( S17A ) , or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cell lysates were immunoprecipitated with anti ‐ LC3 antibody .

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J HeLa cells were transfected with FUNDC1‐Myc, FUNDC1-Myc (S17A), or FUNDC1-Myc (S17D). 24 h after transfection, cell lysates were immunoprecipitated with anti‐LC3 antibody.


A ULK1 + / + or ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 , and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays .

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A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


B ULK1 + / + or ULK1 − / − cells were treated with or without FCCP ( 20 μM ) in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc and then treated with FCCP ( 20 μM ) for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays .

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B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


C ULK1 + / + , ULK1 − / − FUNDC1 ‐ KD , or ULK1 − / − FUNDC1 ‐ KD cells rescued by introduction of HA ‐ ULK1 and FUNDC1 ‐ Myc were cultured under hypoxic ( 1 % O2 ) conditions for 24 h or with FCCP ( 20 μM ) for 24 h . Samples were analyzed by electron microscopy . Scale bar , 2 μm . The yellow asterisks mark mitochondria . The red arrows indicate double ‐ membraned autophagic structures . The yellow arrow denotes autolysosomes .

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C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.


D - E The mitochondrial coverage and mitochondrial − containing AVs shown in ( C ) were quantified . Recon , reconstitution .

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

D-E The mitochondrial coverage and mitochondrial−containing AVs shown in (C) were quantified. Recon, reconstitution.


A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5 ‐ GFP knock ‐ in ( LGR5 ‐ GFPki ) mice . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . B Quantification of GFP staining intensity of intestinal stem and progenitor cells ( ISPCs ) at indicated positions in basal crypts of LGR5 ‐ GFPkimice ( n = 80 crypts from four mice ) . Each dot presents one cell . Red lines : mean values ± SEM . Unpaired two ‐ tailed Student ' s t ‐ test .

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

A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5‐GFP knock‐in (LGR5‐GFPki) mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.B Quantification of GFP staining intensity of intestinal stem and progenitor cells (ISPCs) at indicated positions in basal crypts of LGR5‐GFPkimice (n = 80 crypts from four mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc + / + mice . Dashed line outlines a crypt . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc + / + mice ( n = 150 crypts from three mice ) . Each dot presents one cell . Red lines : mean values ± SEM . Unpaired two ‐ tailed Student ' s t ‐ test .

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

C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc+/+mice. Dashed line outlines a crypt. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc+/+mice (n = 150 crypts from three mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5 + ( GFP ‐ positive ) cell gating .

E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5+ (GFP‐positive) cell gating.


F Representative FACS plots showing gating of LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , and LGR5lo ‐ low populations within the LGR5hi gating .

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

F Representative FACS plots showing gating of LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations within the LGR5hi gating.


G - I mRNA expression of indicated genes relative to GAPDH in LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , and LGR5lo ‐ low populations ( n = 3 mice ) . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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

G-I mRNA expression of indicated genes relative to GAPDH in LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations (n = 3 mice). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A mRNA expression of indicated components of the canonical Wnt / β ‐ catenin pathway in freshly isolated small intestinal crypts of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 5 mice per group ) .

A mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 5 mice per group).


B Axin2 mRNA expression in LGR5 + cells of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and mTerc + / + mice ( n = 3 mice per group ) .

O O O O O O O O O O O O O O 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 Axin2 mRNA expression in LGR5+ cells of 12‐ to 16‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


C mRNA expression of indicated components of the canonical Wnt / β ‐ catenin pathway and of the p53 target gene p21 in cultured crypts of 2 ‐ month ‐ old G3 mTerc − / − mice and mTerc + / + mice ( n = 3 mice per group ) .

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

C mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway and of the p53 target gene p21 in cultured crypts of 2‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


A - J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young ( 2 - 3 month ) and old ( 12 - 16 month ) LGR5 ‐ GFPki , mTerc + / + mice and LGR5 ‐ GFPki , G3 mTerc − / − mice ( n = 4 mice per group ) . ( A , B , D , E , H , I ) Representative FACS plots depicting the analysis of LGR5 + cells . Note the reduction in LGR5 + cells in G3 mTerc − / − mice with the remaining cells showing almost exclusively weak expression of GFP ( LGR5lo ) and that within the fraction of LGR5hi cells , the cells with high LGR5 ‐ reporter activity ( LGR5hi ‐ high ) are preferentially depleted in response to IR . ( C , F ) Quantification of ( C ) the number of LGR5 + cells and ( F ) the number of LGR5hi and LGR5lo cells . ( G ) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5 + cells . ( J ) Quantification of the survival rate of LGR5hi ‐ high cells and LGR5hi ‐ low cells within the fraction of LGR5hi cells comparing old G3 mTerc − / − mice to mTerc + / + mice .

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

A-J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young (2-3 month) and old (12-16 month) LGR5‐GFPki, mTerc+/+mice and LGR5‐GFPki, G3 mTerc−/−mice (n = 4 mice per group). (A, B, D, E, H, I) Representative FACS plots depicting the analysis of LGR5+ cells. Note the reduction in LGR5+ cells in G3 mTerc−/−mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo) and that within the fraction of LGR5hi cells, the cells with high LGR5‐reporter activity (LGR5hi‐high) are preferentially depleted in response to IR. (C, F) Quantification of (C) the number of LGR5+ cells and (F) the number of LGR5hi and LGR5lo cells. (G) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5+ cells. (J) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing old G3 mTerc−/−mice to mTerc+/+mice.


K - O Immunofluorescence microscopy staining of GFP in basal crypts of 9 ‐ month ‐ old LGR5 ‐ GFPki , G3 mTerc − / − mice and age ‐ matched LGR5 ‐ GFPki , mTerc + / + mice ( n = 2 mice per group ) . ( K , L ) Representative pictures are given . Dashed lines outline the crypts . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . ( M ) Quantification of absolute number of GFP + ISPCs at indicated positions at the crypt base per 50 crypts . ( N ) The histogram depicts the depletion rate ( % ) of LGR5 + cells in LGR5 ‐ GFPki , G3 mTerc − / − mice compared to age ‐ matched LGR5 ‐ GFPki , mTerc + / + mice at indicated positions in the basal crypts . ( O ) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts . Note that the staining intensity of position 4 cells is equal in mTerc + / + mice and G3 mTerc − / − mice and is significantly lower than position 1 and 2 cells of mTerc + / + mice .

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

K-O Immunofluorescence microscopy staining of GFP in basal crypts of 9‐month‐old LGR5‐GFPki, G3 mTerc−/−mice and age‐matched LGR5‐GFPki, mTerc+/+mice (n = 2 mice per group). (K, L) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (M) Quantification of absolute number of GFP+ISPCs at indicated positions at the crypt base per 50 crypts. (N) The histogram depicts the depletion rate (%) of LGR5+ cells in LGR5‐GFPki, G3 mTerc−/−mice compared to age‐matched LGR5‐GFPki, mTerc+/+mice at indicated positions in the basal crypts. (O) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts. Note that the staining intensity of position 4 cells is equal in mTerc+/+mice and G3 mTerc−/−mice and is significantly lower than position 1 and 2 cells of mTerc+/+mice.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( A , B ) H & amp ; amp ; E staining , ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of ( C ) spindle ‐ shaped cells in between and adjacent to the Paneth cells . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of (A, B) H&amp;E staining, (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (C) spindle‐shaped cells in between and adjacent to the Paneth cells. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( D , E ) PCNA staining . Arrowheads and numbers indicate the position of ISPCs in the basal crypts . Scale bars : 20 μm . ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for ( F ) PCNA . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 4-5 mice per group). Representative pictures of (D, E) PCNA staining. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for (F) PCNA . Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( ( G , H ) Msi1 staining are given . Arrowheads and numbers indicate the position of ISPCs in the basal crypts . Scale bars : 20 μm . ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of ( I ) Msi1 . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of ((G, H) Msi1 staining are given. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (I) Msi1. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


J , K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9 ‐ month ‐ old G3 mTerc − / − mice ( K ) and age ‐ matched mTerc + / + ( J ) mice . Arrowheads point to positive cells . Dashed lines outline the crypts . Note that the remaining ISPCs in G3 mTerc − / − mice were mostly located in position 4 above the Paneth cells . Scale bar : 20 μm .

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

J, K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9‐month‐old G3 mTerc−/− mice (K) and age‐matched mTerc+/+ (J) mice. Arrowheads point to positive cells. Dashed lines outline the crypts. Note that the remaining ISPCs in G3 mTerc−/− mice were mostly located in position 4 above the Paneth cells. Scale bar: 20 μm.


A - I Three ‐ month ‐ old mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR ( n = 5 mice per group ) . ( A - F ) Representative pictures of PCNA staining . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . Note the depletion of PCNA + CBC cells in position 1 and 2 in between the Paneth cells at 3 - 48 h after IR and the regeneration of these cells at 4 - 6 days after IR . ( G ) Quantification of PCNA + ISPCs at the indicated positions in basal crypts at the indicated time points after IR . Mean values ± SEM are given .

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

A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (A-F) Representative pictures of PCNA staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. Note the depletion of PCNA+ CBC cells in position 1 and 2 in between the Paneth cells at 3-48 h after IR and the regeneration of these cells at 4-6 days after IR. (G) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts at the indicated time points after IR. Mean values ± SEM are given.


A - I Three ‐ month ‐ old mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR ( n = 5 mice per group ) . ( H , I ) Representative pictures of Olfm4 in situ hybridization . Arrowheads point to positive cells . Dashed lines outline the crypts in irradiated samples . Scale bar : 20 μm . Note the selective survival of ISPCs above the Paneth cells at 24 h after IR .

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

A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (H, I) Representative pictures of Olfm4 in situ hybridization. Arrowheads point to positive cells. Dashed lines outline the crypts in irradiated samples. Scale bar: 20 μm. Note the selective survival of ISPCs above the Paneth cells at 24 h after IR.


J - X Three ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR . ( J - N ) Representative pictures of GFP staining at indicated time points after irradiation . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm .

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

J-X Three‐month‐old LGR5‐GFPki, mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (J-N) Representative pictures of GFP staining at indicated time points after irradiation. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.


J - X Three ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR . ( O - X ) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR ( n = 4 - 10 mice per group ) . ( O ) The histogram depicts the number of LGR5 + cells in freshly isolated basal crypts at the indicated time point after IR . ( P , Q ) Flow cytometry analysis was used to determine the percentage of ( P ) LGR5hi cells and ( Q ) LGR5lo cells within the fraction of LGR5 + cells . ( R ‐ W ) Representative FACS plots of small intestinal crypt cells of non ‐ irradiated mice , 24 h after irradiation . Note the reduction in LGR5 + cells in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP ( LGR5lo ) . ( X ) Quantification of the survival rate of LGR5hi ‐ high cells and LGR5hi ‐ low cells within the fraction of LGR5hi cells comparing irradiated mice to non ‐ irradiated mice . Note the preferential depletion of LGR5hi ‐ high cells within LGR5hi cell population upon IR . Mean values ± SEM are given . NIR , non ‐ irradiated ; IR , irradiated .

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

J-X Three‐month‐old LGR5‐GFPki, mTerc+/+mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (O-X) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR (n = 4-10 mice per group). (O) The histogram depicts the number of LGR5+ cells in freshly isolated basal crypts at the indicated time point after IR. (P, Q) Flow cytometry analysis was used to determine the percentage of (P) LGR5hi cells and (Q) LGR5lo cells within the fraction of LGR5+ cells. (R‐W) Representative FACS plots of small intestinal crypt cells of non‐irradiated mice, 24 h after irradiation. Note the reduction in LGR5+ cells in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo). (X) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing irradiated mice to non‐irradiated mice. Note the preferential depletion of LGR5hi‐high cells within LGR5hi cell population upon IR. Mean values ± SEM are given. NIR, non‐irradiated; IR, irradiated.


B , C LGR5hi and LGR5lo cells were freshly isolated from 3 ‐ month ‐ old LGR5 ‐ GFPki mice 6 h after 12 Gy γ ‐ irradiation ( IR ) or from non ‐ irradiated ( NIR ) mice ( n = 3 mice per group ) . ( B ) qPCR analysis of relative mRNA expression of apoptosis ‐ related genes compared to GAPDH . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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

B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (B) qPCR analysis of relative mRNA expression of apoptosis‐related genes compared to GAPDH. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


B , C LGR5hi and LGR5lo cells were freshly isolated from 3 ‐ month ‐ old LGR5 ‐ GFPki mice 6 h after 12 Gy γ ‐ irradiation ( IR ) or from non ‐ irradiated ( NIR ) mice ( n = 3 mice per group ) . ( C ) Representative Western blot analysis of cell lysates for the expression of phospho ‐ p53 and cleaved caspase ‐ 3 .

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

B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (C) Representative Western blot analysis of cell lysates for the expression of phospho‐p53 and cleaved caspase‐3.


D , E Three ‐ month ‐ old mTerc + / + mice were γ ‐ irradiated with 12 Gy . Small intestinal tissue was collected at 3 h after IR ( n = 3 mice per group ) . ( D ) Representative picture of TUNEL staining . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . ( E ) Percentage of TUNEL ‐ positive ISPCs at indicated positions in basal crypts . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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

D, E Three‐month‐old mTerc+/+ mice were γ‐irradiated with 12 Gy. Small intestinal tissue was collected at 3 h after IR (n = 3 mice per group). (D) Representative picture of TUNEL staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (E) Percentage of TUNEL‐positive ISPCs at indicated positions in basal crypts. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


F - P Two ‐ month ‐ old LGR5 ‐ GFPki , p53 + / + mice and LGR5 ‐ GFPki , p53 − / − mice were exposed to 12 Gy γ ‐ irradiation ( n = 4 mice per group ) . Basal crypts were isolated at 24 h after IR . ( F - H ) Flow cytometry analysis of the survival rate of LGR5 + cells ( F ) , LGR5hi and LGR5lo cells ( G ) , and LGR5hi ‐ high and LGR5hi ‐ low cells ( H ) of irradiated mice ( 24 h after IR ) compared to non ‐ irradiated mice ( NIR ) . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test . ( I - P ) Representative FACS plots of LGR5 + cells in freshly isolated basal crypts from irradiated and non ‐ irradiated mice of indicated genotypes . Note the stronger rescue of survival rate of LGR5hi ‐ high than the LGR5hi ‐ low cells upon p53 deletion . NIR , non ‐ irradiated ; IR , irradiated .

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

F-P Two‐month‐old LGR5‐GFPki, p53+/+ mice and LGR5‐GFPki, p53−/− mice were exposed to 12 Gy γ‐irradiation (n = 4 mice per group). Basal crypts were isolated at 24 h after IR. (F-H) Flow cytometry analysis of the survival rate of LGR5+ cells (F), LGR5hi and LGR5lo cells (G), and LGR5hi‐high and LGR5hi‐low cells (H) of irradiated mice (24 h after IR) compared to non‐irradiated mice (NIR). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. (I-P) Representative FACS plots of LGR5+ cells in freshly isolated basal crypts from irradiated and non‐irradiated mice of indicated genotypes. Note the stronger rescue of survival rate of LGR5hi‐high than the LGR5hi‐low cells upon p53 deletion. NIR, non‐irradiated; IR, irradiated.