B ) Total T cells were enriched from spleens of Ptpn6fl / fl / Ptpn11fl / flWbm , CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm , CD4cre Ptpn6fl / fl mice . Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates , Gapdh used as loading control .

O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O

B) Total T cells were enriched from spleens of Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl mice. Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates, Gapdh used as loading control.


CD4cre Ptpn6fl / fl and control mice were subcutaneously injected with MC38 cells . Tumour growth in individual mice is shown for the indicated genotypes and treatments ; number of mice eradicating the tumour is shown within the graphs ( C ) .

B-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Tumour growth in individual mice is shown for the indicated genotypes and treatments; number of mice eradicating the tumour is shown within the graphs (C).


CD4cre Ptpn6fl / fl and control mice were subcutaneously injected with MC38 cells . Survival curves are shown ( D ) .

B-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Survival curves are shown (D).


Tumour growth in individual mice challenged with MC38 and subsequently treated with anti - PD - 1 antibody or isotype control is shown for each indicated genotype ; number of mice eradicating the tumour is shown within the graphs ( G ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

Tumour growth in individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype; number of mice eradicating the tumour is shown within the graphs (G).


individual mice challenged with MC38 and subsequently treated with anti - PD - 1 antibody or isotype control is shown for each indicated genotype Survival curves of CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm and control mice is shown ( H ) .

O B-ORGANISM O O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-ORGANISM O O O O O O

individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype Survival curves of CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm and control mice is shown (H).


I , J ) 10 - 12 days following MC38 tumour inoculation and treatment with anti - PD - 1 antibody or isotype control , CD4cre Ptpn6 / 11wt / wt and CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm mice were sacrificed , and TILs analysed . Graphs depict frequencies of CD8 + T cells ( gated as CD45 + TCRβ + CD8 + ) expressing IFN - γ and TNFα upon re - stimulation ( G ) and percentages of CD8 + T cells ( H ) .

O O O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O

I, J) 10-12 days following MC38 tumour inoculation and treatment with anti-PD-1 antibody or isotype control, CD4cre Ptpn6/11wt/wt and CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm mice were sacrificed, and TILs analysed. Graphs depict frequencies of CD8+ T cells (gated as CD45+ TCRβ+ CD8+) expressing IFN-γ and TNFα upon re-stimulation (G) and percentages of CD8+ T cells (H).


B ) Ptpn6 and Ptpn11 copy number , as estimated through high resolution mass spectrometry in GzmBcre Ptpn6 / 11fl / fl and control CTLs .

O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O

B) Ptpn6 and Ptpn11 copy number, as estimated through high resolution mass spectrometry in GzmBcre Ptpn6/11fl/fl and control CTLs.


C and D ) GzmBcre R26R - YFP reporter mice were injected subcutaneously with MC38 cells and analysed after 11 days . Graphs show the percentage of YFP + cells among CD45 + CD8 + TILs ( C ) and the percentage of PD - 1 + cells among CD45 + CD8 + YFP + TILs ( D ) . Results depict n = 4 mice ( C and D ) .

O O O O B-GENEPROD O O B-GENEPROD O B-ORGANISM O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O

C and D) GzmBcre R26R-YFP reporter mice were injected subcutaneously with MC38 cells and analysed after 11 days. Graphs show the percentage of YFP+ cells among CD45+ CD8+ TILs (C) and the percentage of PD-1+ cells among CD45+ CD8+ YFP+ TILs (D). Results depict n = 4 mice (C and D).


GzmBcre Ptpn6 / 11fl / fl and control Ptpn6 / 11fl / fl mice were subcutaneously injected with MC38 cells . Survival curves and statistical comparisons between different groups are shown ( E ) .

B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

GzmBcre Ptpn6/11fl/fl and control Ptpn6/11fl/fl mice were subcutaneously injected with MC38 cells. Survival curves and statistical comparisons between different groups are shown (E).


The tumour start depicts the time ( days ) following tumour engraftment after which the tumour is palpable , while tumour endpoint depicts the time ( days ) from tumour start until the reaching the maximal allowed tumour size ( F ) .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-EXP_ASSAY I-EXP_ASSAY O O O O

The tumour start depicts the time (days) following tumour engraftment after which the tumour is palpable, while tumour endpoint depicts the time (days) from tumour start until the reaching the maximal allowed tumour size (F).


C ) Numbers of CTLs ( gated on CD8 + T cells ) from Ptpn6 / 11fl / fl or GzmBcre Ptpn6 / 11fl / fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells .

O O O O B-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O

C) Numbers of CTLs (gated on CD8+ T cells) from Ptpn6/11fl/fl or GzmBcre Ptpn6/11fl/fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells.


D ) Quantitative high - resolution mass spectrometry was used to resolve the proteome of Ptpn6 / 11fl / fl and GzmBcre Ptpn6 / 11fl / fl CTLs ( day 7 ) . The plot depicts protein copy number in control and GzmBcre Ptpn6 / 11fl / fl cells . Significantly altered proteins are indicated in red ; selected examples are annotated . Results are based on n = 3 biological replicates and two - tailed , unequal - variance t - test on log10 transformed copy number per cell values was used to compare differences between experimental groups .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

D) Quantitative high-resolution mass spectrometry was used to resolve the proteome of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (day 7). The plot depicts protein copy number in control and GzmBcre Ptpn6/11fl/fl cells. Significantly altered proteins are indicated in red; selected examples are annotated. Results are based on n = 3 biological replicates and two-tailed, unequal-variance t-test on log10 transformed copy number per cell values was used to compare differences between experimental groups.


G ) Graph illustrates the percentage of DAPI + dead cells of Ptpn6 / 11fl / fl and GzmBcre Ptpn6 / 11fl / fl CTLs ( gated on CD8 + T cells ) at the indicated days as measured by flow cytometry .

O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

G) Graph illustrates the percentage of DAPI+ dead cells of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (gated on CD8+ T cells) at the indicated days as measured by flow cytometry.


H ) Protein copy number of Tnfrsf1b and Mcl - 1 in GzmB cre Ptpn6 / 11fl / fl and control CTLs , as estimated through high resolution mass spectrometry .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

H) Protein copy number of Tnfrsf1b and Mcl-1 in GzmB cre Ptpn6/11fl/fl and control CTLs, as estimated through high resolution mass spectrometry.


Representative sections of WT and MPSIIIA brains at 9 months of age stained for astrocytes ( GFAP ) or microglia ( isolectin B4 , ILB4 ) . Images correspond to high power views covering cortical layers II - V . Scale bar , 100 μm . ( n = 4 mice per group ) .

O O O O O B-DISEASE B-TISSUE O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O O

Representative sections of WT and MPSIIIA brains at 9 months of age stained for astrocytes (GFAP) or microglia (isolectin B4, ILB4). Images correspond to high power views covering cortical layers II-V. Scale bar, 100 μm. (n=4 mice per group).


Quantitative PCR for various inflammatory cytokines in whole brains from 9 month old WT and MPSIIIA mice . Symbols above bars are versus WT . ( n = 6 mice per group ) . Data are expressed as mean ± STDEV , data for each cytokine were tested by unpaired t - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus WT .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative PCR for various inflammatory cytokines in whole brains from 9 month old WT and MPSIIIA mice. Symbols above bars are versus WT. (n=6 mice per group). Data are expressed as mean ± STDEV, data for each cytokine were tested by unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus WT.


Quantification of IL - 1β and IL - 1Ra levels in plasma from human healthy control and MPSIII patients ( n = 6 control participants and n = 21 MPSIII patients ) . Data are expressed as mean ± STDEV and were tested by unpaired t - test . C . IL - 1β , control vs . MPSIII P = 0 . 0407 ; D . IL - 1Ra , control vs . MPSIII P = 0 . 0001 . Quantification of IL - 1β and IL - 1Ra levels in cerebrospinal fluid ( CSF ) from control and MPSIII patients ( n = 3 control participants and n = 21 MPSIII patients ) . Data are expressed as mean ± STDEV and were tested by unpaired t - test ; * P < 0 . 05 . F . IL - 1Ra , control vs . MPSIII P = 0 . 0167 .

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-ORGANISM O O O B-DISEASE O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-DISEASE O O O O O O O O O O O B-DISEASE O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O O O O

Quantification of IL-1β and IL-1Ra levels in plasma from human healthy control and MPSIII patients (n=6 control participants and n=21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test. C. IL-1β, control vs. MPSIII P=0.0407; D. IL-1Ra, control vs. MPSIII P=0.0001. Quantification of IL-1β and IL-1Ra levels in cerebrospinal fluid (CSF) from control and MPSIII patients (n=3 control participants and n=21 MPSIII patients). Data are expressed as mean ± STDEV and were tested by unpaired t-test; *P<0.05. F. IL-1Ra, control vs. MPSIII P=0.0167.


The expression of Il1b was measured in whole brain using quantitative PCR 1 hour , 2 hours and 6 hours after I . P . challenge ( n = 6 mice per group ) . Data are expressed as mean ± STDEV and were tested by two - way ANOVA with Bonferroni ' s post - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus PBS at the relevant time - point .

O B-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The expression of Il1b was measured in whole brain using quantitative PCR 1 hour, 2 hours and 6 hours after I.P. challenge (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus PBS at the relevant time-point.


Quantification of intracellular production of IL - 1β ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * * * P < 0 . 001 . Symbols above bars are versus PBS . Quantification of IL - 1β secreted into the media ( n = 3 independent experiments each with 3 inter - experimental replicates ) . A horizontal dotted line represents the limit of detection of the ELISA . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus PBS .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of intracellular production of IL-1β (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus PBS. Quantification of IL-1β secreted into the media (n=3 independent experiments each with 3 inter-experimental replicates). A horizontal dotted line represents the limit of detection of the ELISA. Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P<0.01, ***P<0.001. Symbols above bars are versus PBS.


WT mixed glial cultures were co - treated with MPSIIIA GAG ( 4 μg / ml ) and / or the TLR4 inhibitor CLI - 095 ( 1 μg / ml ) for 24 hours . The intracellular production of IL - 1β was measured ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * * * P < 0 . 001 . Symbols above bars are versus MPSIIIA GAG + vehicle .

O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O

WT mixed glial cultures were co-treated with MPSIIIA GAG (4 μg/ml) and/or the TLR4 inhibitor CLI-095 (1 μg/ml) for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus MPSIIIA GAG + vehicle.


Multi - heparinase ( HepM ) and / or chondroitinase ABC ( cABC ) digests were performed on MPSIIIA GAG , and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours alongside heparin , bovine HS ( bHS ) , porcine DS ( pDS ) and WT GAG . The intracellular production of IL - 1β was measured ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * P < 0 . 05 , * * * P < 0 . 001 . Symbols above bars are versus MPSIIIA GAG alone .

B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-DISEASE B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O

Multi-heparinase (HepM) and/or chondroitinase ABC (cABC) digests were performed on MPSIIIA GAG, and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours alongside heparin, bovine HS (bHS), porcine DS (pDS) and WT GAG. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001. Symbols above bars are versus MPSIIIA GAG alone.


MPSIIIA GAG was 2 - O - desulphated ( hashed bars ) utilising 120 mM NaOH , and compositional HS disaccharide analysis performed via RP - HPLC against untreated MPSIIIA GAG ( black bars ) to validate complete 2 - O - desulphation . Percentage contribution of each modification were determined ( n = 3 GAG samples ) . Data are expressed as mean ± STDEV and were tested by two - way ANOVA with Bonferroni ' s post - test ; 2S MPSIIIA GAG vs . 2S 2 - ODS MPSIIIA GAG P < 0 . 0001 . NS , N - sulphated glucosamine ; 2S , 2 - O - sulphate group ; 6S , 6 - O sulphate group .

B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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-SMALL_MOLECULE O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

MPSIIIA GAG was 2-O-desulphated (hashed bars) utilising 120 mM NaOH, and compositional HS disaccharide analysis performed via RP-HPLC against untreated MPSIIIA GAG (black bars) to validate complete 2-O-desulphation. Percentage contribution of each modification were determined (n=3 GAG samples). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; 2S MPSIIIA GAG vs. 2S 2-ODS MPSIIIA GAG P<0.0001. NS, N-sulphated glucosamine; 2S, 2-O-sulphate group; 6S, 6-O sulphate group.


MPSIIIA GAG was 2 - O - desulphated and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours . The intracellular production of IL - 1β was measured ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; MPSIIIA GAG vs . 2ODS MPSIIIA GAG P < 0 . 0001 . 2ODS , 2 - O - desulphated .

B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O

MPSIIIA GAG was 2-O-desulphated and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; MPSIIIA GAG vs. 2ODS MPSIIIA GAG P<0.0001. 2ODS, 2-O-desulphated.


Representative sections from 9 month old WT and MPSIIIA brains showing secondary storage of GM2 ganglioside , cholesterol , protein aggregates and amyloid beta peptide 1 - 40 and 1 - 42 within cortical layers II to V / VI , x20 , scale bar : 50 μm . Inserts , 100x , scale bar : 10μm . ( n = 4 mice per group )

O O O O O O O O B-DISEASE B-TISSUE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE I-TISSUE O O 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-ORGANISM O O O

Representative sections from 9 month old WT and MPSIIIA brains showing secondary storage of GM2 ganglioside, cholesterol, protein aggregates and amyloid beta peptide 1-40 and 1-42 within cortical layers II to V/VI, x20, scale bar: 50 μm. Inserts, 100x, scale bar: 10μm. (n=4 mice per group)


ATP levels in whole brain homogenate from 9 month old WT and MPSIIIA mice ( n = 6 mice per group ) . Data are expressed as mean ± STDEV and were tested by unpaired t - test . WT vs . MPSIIIA P < 0 .

B-SMALL_MOLECULE O O O B-TISSUE O O O O O O O B-DISEASE B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O

ATP levels in whole brain homogenate from 9 month old WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.02


Brain transcription of mRNA species for various inflammasome components ( n = 6 mice per group ) . Data are expressed as mean ± STDEV , data for each cytokine were tested by unpaired t - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus

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Brain transcription of mRNA species for various inflammasome components (n=6 mice per group). Data are expressed as mean ± STDEV, data for each cytokine were tested by unpaired t-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus WT


Caspase 1 activity levels in whole brain homogenate from WT and MPSIIIA mice ( n = 6 mice per group ) . Data are expressed as mean ± STDEV and were tested by unpaired t - test . WT vs . MPSIIIA P < 0 .

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Caspase 1 activity levels in whole brain homogenate from WT and MPSIIIA mice (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by unpaired t-test. WT vs. MPSIIIA P<0.0001


WT ( E - G ) and Nlrp3 - / - ( H - J ) mixed glia were primed with MPSIIIA GAG or LPS . ATP ( 5 mM ) , cholesterol crystals ( 1 mg / ml ) or amyloid - β fibrils ( Aβ1 - 40 4 . 3 μM and Aβ1 - 42 5 μM ) were added 4 hours post - priming stimulus , and the secretion of IL - 1β into the media measured via ELISA after a further 20 hours ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * P < 0 . 05 , * * * P < 0 .

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WT (E-G) and Nlrp3-/- (H-J) mixed glia were primed with MPSIIIA GAG or LPS. ATP (5 mM), cholesterol crystals (1 mg/ml) or amyloid-β fibrils (Aβ1-40 4.3 μM and Aβ1-42 5 μM) were added 4 hours post-priming stimulus, and the secretion of IL-1β into the media measured via ELISA after a further 20 hours (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001


The biological activity of LV - mediated IL - 1Ra was assessed in vitro . RAW264 . 7 macrophages were transduced with LV . IL1RN or LV . GFP vectors at a MOI of 10 . After 72 hours cells were stimulated with recombinant hIL - 1β and the expression of TNF - α assessed after a further 48 hours . ( n = 3 independent experiments each with 3 inter - experimental replicates ) . Data are expressed as mean ± STDEV and were tested by two - way ANOVA with Bonferroni ' s post - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus NTC within a treatment group .

O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The biological activity of LV-mediated IL-1Ra was assessed in vitro. RAW264.7 macrophages were transduced with LV.IL1RN or LV.GFP vectors at a MOI of 10. After 72 hours cells were stimulated with recombinant hIL-1β and the expression of TNF-α assessed after a further 48 hours. (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus NTC within a treatment group.


A portion of transduced HSCs were plated into CFU assays , incubated for 12 days and CFU colonies analysed . Vector copy number were quantified ( n = 6 CFU assays ) .

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A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. Vector copy number were quantified (n=6 CFU assays).


A portion of transduced HSCs were plated into CFU assays , incubated for 12 days and CFU colonies analysed . protein production of secreted human IL - 1Ra were quantified ( n = 6 CFU assays ) .

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A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. protein production of secreted human IL-1Ra were quantified (n=6 CFU assays).


A portion of transduced HSCs were plated into CFU assays , incubated for 12 days and CFU colonies analysed . intracellular human IL - 1Ra were quantified ( n = 6 CFU assays ) .

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A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. intracellular human IL-1Ra were quantified (n=6 CFU assays).


Donor chimerism was determined by flow cytometry at 4 months of age ( 2 months post - transplant ) ( n = 10 mice ) .

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Donor chimerism was determined by flow cytometry at 4 months of age (2 months post-transplant) (n=10 mice).


based on peripheral blood composition was determined by flow cytometry at 4 months of age ( 2 months post - transplant ) ( n = 10 mice ) .

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based on peripheral blood composition was determined by flow cytometry at 4 months of age (2 months post-transplant) (n=10 mice).


Human IL - 1Ra expression was quantified in the plasma of mice at 6 months of age ( 4 months post - transplant ) ( n = 10 mice per group ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; WT vs . LV . IL1RN P < 0 . 0001 ; MPSIIIA vs . LV . IL1RN P < 0 . 0001 . Symbols above bars are versus WT .

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Human IL-1Ra expression was quantified in the plasma of mice at 6 months of age (4 months post-transplant) (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT.


Vector copy number was evaluated in brain , spleen , liver , white blood cells ( WBC ) and bone marrow ( BM ) of mice at 6 months of age ( 4 months post - transplant ) ( n = 10 mice per group ) .

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Vector copy number was evaluated in brain, spleen, liver, white blood cells (WBC) and bone marrow (BM) of mice at 6 months of age (4 months post-transplant) (n=10 mice per group).


Quantification of human IL - 1Ra levels in the brain of transplanted MPSIIIA mice ( n = 10 mice per group ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; WT vs . LV . IL1RN P < 0 . 0001 ; MPSIIIA vs . LV . IL1RN P < 0 . 0001 . Symbols above bars are versus WT .

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Quantification of human IL-1Ra levels in the brain of transplanted MPSIIIA mice (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; WT vs. LV.IL1RN P<0.0001; MPSIIIA vs. LV.IL1RN P<0.0001. Symbols above bars are versus WT.


Representative sections from 6 - month old MPSIIIA and LV . IL1RN transplanted MPSIIIA mice . Sections show the CA3 region of the hippocampus . Cells expressing high levels of human IL - 1Ra are indicated with black arrows . 40x , scale bar , 50 μm ( n = 4 mice per group ) .

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Representative sections from 6-month old MPSIIIA and LV.IL1RN transplanted MPSIIIA mice. Sections show the CA3 region of the hippocampus. Cells expressing high levels of human IL-1Ra are indicated with black arrows. 40x, scale bar, 50 μm (n=4 mice per group).


Mice were placed in the centre of the Y - maze and allowed to explore freely for 10 minutes . The percentage of spontaneous alternations in the Y - maze were measured to assess working memory ( n = 10 mice per group ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * P < 0 . 05 , * * P < 0 .

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Mice were placed in the centre of the Y-maze and allowed to explore freely for 10 minutes. The percentage of spontaneous alternations in the Y-maze were measured to assess working memory (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, **P<0.01


A one - hour open field test was performed and path length measured as an indicator of hyperactivity ( n = 10 mice per group ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * * * P < 0 . 001 . Symbols above bars are versus

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A one-hour open field test was performed and path length measured as an indicator of hyperactivity (n=10 mice per group). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus WT


Representative images of the hippocampus and cortex ( layers II - V ) from control and treated mice stained with anti - mouse glial fibrillary acidic protein ( GFAP ; red ) / DAPI ( nuclear , blue ) to identify activated astrocytes , 20x , scale bar : 50μm Representative images of the hippocampus and cortex ( layers II - V ) from control and treated mice stained with isolectin B4 ( ILB4 ) to identify activated microglia , 20x , scale bar :

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Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with anti-mouse glial fibrillary acidic protein (GFAP; red)/DAPI (nuclear, blue) to identify activated astrocytes, 20x, scale bar: 50μm Representative images of the hippocampus and cortex (layers II-V) from control and treated mice stained with isolectin B4 (ILB4) to identify activated microglia, 20x, scale bar: 50μ


The average number of GFAP positive astrocyte cells over three fields of view per mouse of the hippocampus ( C ) and the cortex ( D ) were counted ( n = 4 mice per group , average of 3 fields of view per mouse ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * * P < 0 . 01 , * * * P < 0 . 001 . Symbols above bars are versus

O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM O O B-TISSUE O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The average number of GFAP positive astrocyte cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P<0.01, ***P<0.001. Symbols above bars are versus WT


The average number of ILB4 + positive microglial cells over three fields of view per mouse of the hippocampus ( C ) and the cortex ( D ) were counted ( n = 4 mice per group , average of 3 fields of view per mouse ) . Data are expressed as mean ± STDEV and were tested by one - way ANOVA with Tukey ' s post - test ; * * * P < 0 . 001 . Symbols above bars are versus

O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-ORGANISM O O B-TISSUE O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The average number of ILB4+ positive microglial cells over three fields of view per mouse of the hippocampus (C) and the cortex (D) were counted (n=4 mice per group, average of 3 fields of view per mouse). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus WT


Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes ( D ) Binding is reduced in Delta ∆ β1 - 2 , but not to the Serrate equivalent using liposomes composed of PC : PS : PE - fluoroscein ( 80 : 15 : 5 ) . Notch binding to Drosophila Delta NE3 ( E ) WT and ∆ β1 - 2 ( Delta ∆ β1 - 2 ) variants both bind to Notch , unlike variant with F204A substitution in DSL domain . Comparisons were performed with a two - tailed unpaired t - test . Values are shown as scattered data points with the dark lines representing the means . ns , no significant difference , * * P < 0 . 01 ; * * * P < 0 . 0001 .

O B-ORGANISM B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes (D) Binding is reduced in Delta∆β1-2, but not to the Serrate equivalent using liposomes composed of PC:PS:PE-fluoroscein (80:15:5). Notch binding to Drosophila Delta NE3 (E) WT and ∆β1-2 (Delta∆β1-2) variants both bind to Notch, unlike variant with F204A substitution in DSL domain. Comparisons were performed with a two-tailed unpaired t-test. Values are shown as scattered data points with the dark lines representing the means. ns, no significant difference, **P < 0.01; ***P < 0.0001.


Adult wings from Dl ∆ β1 - 2 flies . No defects are detected in wings from Dl ∆ β1 - 2 / + ( B ) , Homozygous Dl ∆ β1 - 2 / Dl ∆ β1 - 2 have extra vein tissue near L5 and uneven L2 veins ( arrowheads ; C ) .

O B-TISSUE O B-GENEPROD O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O O O O O O

Adult wings from Dl∆β1-2 flies. No defects are detected in wings from Dl∆β1-2/+ (B), Homozygous Dl∆β1-2/ Dl∆β1-2 have extra vein tissue near L5 and uneven L2 veins (arrowheads; C).


Microchaetae are arranged in rows on the thorax of control ( yw ) flies ; these become disordered and more dense in Dl ∆ β1 - 2 / Dl ∆ β1 - 2 . White rectangle indicates area scored for E Number of microchaetes per central area ( white rectangle in D ) in the indicated genotypes .

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Microchaetae are arranged in rows on the thorax of control (yw) flies; these become disordered and more dense in Dl∆β1-2/Dl∆β1-2. White rectangle indicates area scored for E Number of microchaetes per central area (white rectangle in D) in the indicated genotypes.


Representative images of adult females wings in combinations of Dl ∆ β1 - 2 with loss of function Delta alleles . In combinations with Dlrev10 ( A ) , Dl ∆ Exon2 ( B ) , or Df ( 3R ) DlFx3 ( C ) , vein thickening is strongly enhanced ( right panels ) compared to heterozygous mutants alone ( left panels ) . Vertical square brackets indicate the regions used for vein thickness quantification . Quantification of wing - vein thickness in females of the indicated genotypes . Representative images of adult female wings demonstrate that Dl ∆ β1 - 2 rescues the wing notching phenotype , caused by a Notch loss of function allele ( N55e11 ) . Horizontal square bracket indicates the L5 vein " delta " at the intersection with the wing margin analyzed in EV2E . Quantification of wing notching in females of the indicated genotypes , Dl ∆ β1 - 2 rescues notching in a similar manner to Dlrev10 .

O O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE O O 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-TISSUE O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O

Representative images of adult females wings in combinations of Dl∆β1-2 with loss of function Delta alleles. In combinations with Dlrev10 (A), Dl∆Exon2 (B), or Df(3R)DlFx3 (C), vein thickening is strongly enhanced (right panels) compared to heterozygous mutants alone (left panels). Vertical square brackets indicate the regions used for vein thickness quantification. Quantification of wing-vein thickness in females of the indicated genotypes. Representative images of adult female wings demonstrate that Dl∆β1-2 rescues the wing notching phenotype, caused by a Notch loss of function allele (N55e11). Horizontal square bracket indicates the L5 vein "delta" at the intersection with the wing margin analyzed in EV2E. Quantification of wing notching in females of the indicated genotypes, Dl∆β1-2 rescues notching in a similar manner to Dlrev10.


Equatorial region of eye imaginal discs where E ( spl ) mδ0 . 5 expression ( green ) becomes restricted to a single photoreceptor in each cluster ( magenta ) , as detected in control and Dl ∆ β1 - 2 / + discs ( top panels ) . In Df ( 3R ) DlFx3 / + and Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 discs ( bottom panels ) E ( spl ) mδ0 . 5 expression is reduced ( Df ( 3R ) DlFx3 / + ) or absent from several clusters ( Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 ) indicative of reduced Notch signalling . Scale bars correspond to 10 μm .

O O O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O

Equatorial region of eye imaginal discs where E(spl)mδ0.5 expression (green) becomes restricted to a single photoreceptor in each cluster (magenta), as detected in control and Dl∆β1-2/+ discs (top panels). In Df(3R)DlFx3 /+ and Dl∆β1-2/Df(3R)DlFx3 discs (bottom panels) E(spl)mδ0.5 expression is reduced (Df(3R)DlFx3 /+) or absent from several clusters (Dl∆β1-2/Df(3R)DlFx3) indicative of reduced Notch signalling. Scale bars correspond to 10 μm.


Proportion of photoreceptors clusters that fail to express the E ( spl ) mδ0 . 5 reporter in the indicated genotypes . ns , no significant difference , * * * P < 0 . 0001 ( one - way ANOVA ) . On the violin plot , dashed line represents the median and the dotted lines show the quartiles .

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Proportion of photoreceptors clusters that fail to express the E(spl)mδ0.5 reporter in the indicated genotypes. ns, no significant difference, *** P<0.0001 (one-way ANOVA). On the violin plot, dashed line represents the median and the dotted lines show the quartiles.


Apical view of wing imaginal disc with homozygous Dl ∆ β1 - 2 clones ( GFP negative ) stained for Dl ( red ) and Cadherin ( blue ) . ( A ' ) Z - projection of apical layers spanning a Dl ∆ β1 - 2 clone ( GFP negative ) located at the DV boundary . No change in apical localisation of Dl ( red ) and Cadherin ( blue ) is detected . Panels on the right show the Dl ( grayscale ) apical localisation on the wing imaginal disc .

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Apical view of wing imaginal disc with homozygous Dl∆β1-2 clones (GFP negative) stained for Dl (red) and Cadherin (blue). (A') Z-projection of apical layers spanning a Dl∆β1-2 clone (GFP negative) located at the DV boundary. No change in apical localisation of Dl (red) and Cadherin (blue) is detected. Panels on the right show the Dl (grayscale) apical localisation on the wing imaginal disc.


Uptake assay at t = 0 . After exposure to extracellular anti - Dl antibody , Dl protein ( red ) is detected at similar levels apical to Cadherin ( blue ) in wild - type ( GFP ) and Dl ∆ β1 - 2 mutant tissue ( GFP negative ) . ( B ' ) Cross - sectional view of B , Dl protein ( red ) is present apically relative to Cadherin ( Blue ) which marks adherens junctions . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively . Uptake assay after 30 minutes , internalised anti - Dl ( red ) enters the endocytic route and in the cross sectional view ( C ' ) can similarly be detected as puncta along the cell axis in wild - type ( GFP ) and homozygous Dl ∆ β1 - 2 ( GFP negative ) tissue . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O

Uptake assay at t=0. After exposure to extracellular anti-Dl antibody, Dl protein (red) is detected at similar levels apical to Cadherin (blue) in wild-type (GFP) and Dl∆β1-2 mutant tissue (GFP negative). (B') Cross-sectional view of B, Dl protein (red) is present apically relative to Cadherin (Blue) which marks adherens junctions. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively. Uptake assay after 30 minutes, internalised anti-Dl (red) enters the endocytic route and in the cross sectional view (C') can similarly be detected as puncta along the cell axis in wild-type (GFP) and homozygous Dl∆β1-2 (GFP negative) tissue. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.


( a ) Western blotting was performed to determine FoxO1 expression , p62 degradation and LC3 - II accumulation in H1299 , HCT116 and HeLa cells in response to serum starvation ( SS ) for 24 h or 0 . 5 mM H2O2 for 6 h in the presence or absence of E64 and pepstatin A ( PEPS A ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

(a) Western blotting was performed to determine FoxO1 expression, p62 degradation and LC3-II accumulation in H1299, HCT116 and HeLa cells in response to serum starvation (SS) for 24 h or 0.5 mM H2O2 for 6 h in the presence or absence of E64 and pepstatin A (PEPS A).


( b , c ) Stable FoxO1 knockdown HCT116 cells , or cells with an empty RNAi plasmid , were incubated in a serum - free medium for up to 36 h ( b ) or treated with 0 . 5 mM H2O2 for up to 12 h ( c ) ; western blotting was performed to detect changes in FoxO1 , p62 , LC3 - II or β - actin .

O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O

(b, c) Stable FoxO1 knockdown HCT116 cells, or cells with an empty RNAi plasmid, were incubated in a serum-free medium for up to 36 h (b) or treated with 0.5 mM H2O2 for up to 12 h (c); western blotting was performed to detect changes in FoxO1, p62, LC3-II or β-actin.


( d ) HCT116 cells were labelled with [ 3H ] leucine for 48 h , washed and incubated for 24 h , before being subjected to serum starvation for 24 h or 0 . 5 mM H2O2 for 12 h in the presence or absence of 3 - MA . At 12 or 24 h after treatment , the relative degradation of long - lived proteins was measured by counting radioactivity . Data in d are means ± s . d . ( n = 3 ) . Two asterisks , P 0 . 01 ; asterisk , P 0 . 05 ; NS , not significant , P > 0 . 05 ; Student ' s t - test .

O O O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HCT116 cells were labelled with [3H]leucine for 48 h, washed and incubated for 24 h, before being subjected to serum starvation for 24 h or 0.5 mM H2O2 for 12 h in the presence or absence of 3-MA. At 12 or 24 h after treatment, the relative degradation of long-lived proteins was measured by counting radioactivity. Data in d are means ± s.d. (n = 3). Two asterisks, P 0.01; asterisk, P 0.05; NS, not significant, P > 0.05; Student's t-test.


( e ) Autophagic vesicles or autophagosomes in HCT116 cells or stable FoxO1 - RNAi HCT116 cells , were observed by electron microscopy after 24 h of serum starvation . The bottom picture enlargement showing clearer autophagic vesicles indicated by an arrow . The graph shows a statistical analysis of autophagic vesicles in HCT116 cells with or without knockdown of FoxO1 in response to serum starvation . Data in e are means ± s . d . ( n = 3 ) .

O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-CELL_LINE O O O B-GENEPROD O O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(e) Autophagic vesicles or autophagosomes in HCT116 cells or stable FoxO1-RNAi HCT116 cells, were observed by electron microscopy after 24 h of serum starvation. The bottom picture enlargement showing clearer autophagic vesicles indicated by an arrow. The graph shows a statistical analysis of autophagic vesicles in HCT116 cells with or without knockdown of FoxO1 in response to serum starvation. Data in e are means ± s.d. (n = 3).


( f ) LC3 - immunopositive punctate signals were observed in HCT116 or in stable FoxO1 - RNAi HCT116 cells in response to serum starvation for 24 h or 0 . 5 mM H2O2 treatment . ( g ) Quantification of LC3 - positive punctate cells in f . Data in g are means ± s . d . ( n = 3 ) .

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(f) LC3-immunopositive punctate signals were observed in HCT116 or in stable FoxO1-RNAi HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 treatment. (g) Quantification of LC3-positive punctate cells in f. Data in g are means ± s. d. (n = 3).


( h ) A rescue experiment using a specific mutated FoxO1 plasmid to validate the activity of FoxO1 in the induction of autophagy in HCT116 cells in the presence or absence of serum , with or without E64 and pepstatin A .

O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O

(h) A rescue experiment using a specific mutated FoxO1 plasmid to validate the activity of FoxO1 in the induction of autophagy in HCT116 cells in the presence or absence of serum, with or without E64 and pepstatin A.


( a ) A Flag - tagged FoxO1 ( WT ) , FoxO1 ( ΔDB ) , FoxO1 ( ΔDB3A ) or an empty plasmid was individually transfected into GFP - LC3 pre - transfected H1299 cells , and the cellular localization of FoxO1 ( upper panels ) or GFP - LC3 punctate signals ( lower panels ) was observed with a confocal microscope . Scale bars , 10 μm . ( b ) The numbers of punctate GFP - LC3 cells are represented by the ratio of GFP - LC3 - labelled punctate cells to overall survived H1299 cells ( at least 200 cells were counted ) . Data in b are means ± s . d . ( n = 3 ) .

O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O 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) A Flag-tagged FoxO1(WT), FoxO1(ΔDB), FoxO1(ΔDB3A) or an empty plasmid was individually transfected into GFP-LC3 pre-transfected H1299 cells, and the cellular localization of FoxO1 (upper panels) or GFP-LC3 punctate signals (lower panels) was observed with a confocal microscope. Scale bars, 10 μm. (b) The numbers of punctate GFP-LC3 cells are represented by the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells (at least 200 cells were counted). Data in b are means ± s.d. (n = 3).


( c ) p62 degradation and LC3 - II accumulation were evaluated by western blotting in H1299 cells transfected with exogenous FoxO1 - expressing plasmids mentioned above .

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(c) p62 degradation and LC3-II accumulation were evaluated by western blotting in H1299 cells transfected with exogenous FoxO1-expressing plasmids mentioned above.


( d ) H1299 cells were labelled with [ 3H ] leucine for 48 h , then transfected with a control plasmid or various Flag - FoxO1 plasmids , in the presence or absence of 3 - MA or E64 and pepstatin A . Degradation of long - lived proteins was measured at the indicated time , 24 h after transfection . Data in d are means ± s . d . ( n = 3 ) .

O O O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O 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) H1299 cells were labelled with [3H]leucine for 48 h, then transfected with a control plasmid or various Flag-FoxO1 plasmids, in the presence or absence of 3-MA or E64 and pepstatin A. Degradation of long-lived proteins was measured at the indicated time, 24 h after transfection. Data in d are means ± s.d. (n = 3).


( e ) A GFP - tagged FoxO1 ( 3A ) or an empty plasmid was co - transfected with a FoxO1 - promoter luciferase plasmid into H1299 cells . After 24 h , luciferase reporter activity of FoxO1 was measured . RLU , relative luciferase units . Data are means ± s . d . ( n = 3 ) .

O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(e) A GFP-tagged FoxO1(3A) or an empty plasmid was co-transfected with a FoxO1-promoter luciferase plasmid into H1299 cells. After 24 h, luciferase reporter activity of FoxO1 was measured. RLU, relative luciferase units. Data are means ± s.d. (n = 3).


( f ) A set of RNAi - resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1 - RNAi HCT116 cells in the presence or absence of E64 and pepstatin A . Western blotting was performed to detect p62 degradation and LC3 - II accumulation .

O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O

(f) A set of RNAi-resistant rescue forms of FoxO1 plasmids were transfected into stable FoxO1-RNAi HCT116 cells in the presence or absence of E64 and pepstatin A. Western blotting was performed to detect p62 degradation and LC3-II accumulation.


( g ) Flag - tagged FoxO1 ( WT ) r , FoxO1 ( 3A ) r , FoxO1 ( 3A - NLSm ) r , FoxO1 ( NESm ) r or an empty plasmid were individually transfected into GFP - LC3 pre - transfected HCT116 cells ( FoxO1 - RNAi treated ) , and the cellular localization of FoxO1 ( upper panels ) or GFP - LC3 punctate signals ( lower panels ) were observed with a confocal microscope . ( h ) The numbers of punctate GFP - LC3 cells are represented by the ratio of GFP - LC3 - labelled punctate cells to overall survived HCT116 cells . Data in h are means ± s . d . ( n = 3 ) .

O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O

(g) Flag-tagged FoxO1(WT)r, FoxO1(3A)r, FoxO1(3A-NLSm)r, FoxO1(NESm)r or an empty plasmid were individually transfected into GFP-LC3 pre-transfected HCT116 cells (FoxO1-RNAi treated), and the cellular localization of FoxO1 (upper panels) or GFP-LC3 punctate signals (lower panels) were observed with a confocal microscope. (h) The numbers of punctate GFP-LC3 cells are represented by the ratio of GFP-LC3-labelled punctate cells to overall survived HCT116 cells. Data in h are means ± s.d. (n = 3).


( a ) Different FoxO1 - expressing plasmids , namely FoxO1 ( WT ) , FoxO1 ( 3A ) , FoxO1 ( ΔDB ) , FoxO1 ( ΔDB3A ) , FoxO1 ( NESm ) and FoxO1 ( 3A - NLSm ) , were transfected into H1299 cells and cell fractionation was performed to analyse cellular localization ( N , nucleus ; C , cytoplasm ) of these FoxO1 mutants . PCAF is a loading control for nuclear protein , and actin is the loading control for cytoplasmic protein .

O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O

(a) Different FoxO1-expressing plasmids, namely FoxO1(WT), FoxO1(3A), FoxO1(ΔDB), FoxO1(ΔDB3A), FoxO1(NESm) and FoxO1(3A-NLSm), were transfected into H1299 cells and cell fractionation was performed to analyse cellular localization (N, nucleus; C, cytoplasm) of these FoxO1 mutants. PCAF is a loading control for nuclear protein, and actin is the loading control for cytoplasmic protein.


( b ) Quantitative PCR was performed to measure the transactivity of autophagy - related genes in transfected H1299 cells with different FoxO1 - expressing plasmids ( upper panel ) or in stable FoxO1 - RNAi or non - RNAi - treated HCT116 cells in response to serum starvation for 24 h or 0 . 5 mM H2O2 for 12 h ( lower panel ) . All data are means ± s . d . ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(b) Quantitative PCR was performed to measure the transactivity of autophagy-related genes in transfected H1299 cells with different FoxO1-expressing plasmids (upper panel) or in stable FoxO1-RNAi or non-RNAi-treated HCT116 cells in response to serum starvation for 24 h or 0.5 mM H2O2 for 12 h (lower panel). All data are means ± s.d. (n = 3).


( c ) Different FoxO1 - mutant - expressing plasmids were transfected into H1299 cells and a ChIP assay was performed to measure the binding activity of these mutated FoxO1 to the p27 promoter .

O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O

(c) Different FoxO1-mutant-expressing plasmids were transfected into H1299 cells and a ChIP assay was performed to measure the binding activity of these mutated FoxO1 to the p27 promoter.


( a ) HCT116 cells were treated with serum starvation for 24 h ( upper panels ) or 0 . 5 mM H2O2 for 12 h ( lower panels ) ; cytosolic proteins were extracted for co - immunoprecipitation with anti - FoxO1 and probed with anti - acetylated lysine ( Ace - lyn ) . Both bottom panels display cytosolic FoxO1 . IP , immunoprecipitation .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O

(a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.5 mM H2O2 for 12 h (lower panels); cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 and probed with anti-acetylated lysine (Ace-lyn). Both bottom panels display cytosolic FoxO1. IP, immunoprecipitation.


( b ) HCT116 cells were serum - starved for 24 h and the cytosolic proteins were extracted for co - immunoprecipitation with anti - FoxO1 or anti - SIRT2 , followed by probing with anti - SIRT2 or anti - FoxO1 , respectively ( top and middle panels ) . FoxO1 ( WT ) was also transfected into H1299 cells and the cytosolic protein was extracted , followed by co - immunoprecipitation with anti - FoxO1 and probing with anti - SIRT2 ( bottom panels ) .

O O O B-CELL_LINE O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O

(b) HCT116 cells were serum-starved for 24 h and the cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 or anti-SIRT2, followed by probing with anti-SIRT2 or anti-FoxO1, respectively (top and middle panels). FoxO1(WT) was also transfected into H1299 cells and the cytosolic protein was extracted, followed by co-immunoprecipitation with anti-FoxO1 and probing with anti-SIRT2 (bottom panels).


( c ) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid . At 48 h after transfection , the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 ( upper panels ) , or for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine ( lower panels ) . IB , immunoblot .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O

(c) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid. At 48 h after transfection, the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 (upper panels), or for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine (lower panels). IB, immunoblot.


( d ) HCT116 cells were treated with AGK2 for 24 h at different doses , as indicated . The cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine .

O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O

(d) HCT116 cells were treated with AGK2 for 24 h at different doses, as indicated. The cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine.


( a , b ) H1299 cells were transfected with an empty control plasmid or with the Flag - FoxO1 ( WT ) plasmid . At 24 h after transfection , the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - mTOR , Beclin 1 , Vps34 or Atg1 ( a ) and with Atg3 , Atg4 , Atg10 , Atg7 or LC3 ( b ) .

O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

(a, b) H1299 cells were transfected with an empty control plasmid or with the Flag-FoxO1(WT) plasmid. At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-mTOR, Beclin 1, Vps34 or Atg1 (a) and with Atg3, Atg4, Atg10, Atg7 or LC3 (b).


( c ) H1299 cells were co - transfected with Myc - Atg7 , with Flag - FoxO1 ( WT ) or with an empty control plasmid . At 24 h after transfection , the cytosolic lysate was extracted for Flag with anti - Flag , followed by probing with anti - Myc .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O

(c) H1299 cells were co-transfected with Myc-Atg7, with Flag-FoxO1(WT) or with an empty control plasmid. At 24 h after transfection, the cytosolic lysate was extracted for Flag with anti-Flag, followed by probing with anti-Myc.


HCT116 cells were serum - starved for 24 h ( d ) and the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

B-CELL_LINE O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O

HCT116 cells were serum-starved for 24 h (d)and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( e ) Different Myc - tagged FoxO1 fragments and Flag - tagged Atg7 were co - transfected into H1299 cells , and the cytosolic lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-CELL_LINE O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O

(e) Different Myc-tagged FoxO1 fragments and Flag-tagged Atg7 were co-transfected into H1299 cells, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) HCT116 cells were transfected with an empty control plasmid , an SIRT2 ( WT ) - expressing plasmid or an SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , cells were serum - starved for 24 h , and the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - Atg7 .

O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD 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-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O

(f) HCT116 cells were transfected with an empty control plasmid, an SIRT2(WT)-expressing plasmid or an SIRT2(168A)-expressing plasmid. At 24 h after transfection, cells were serum-starved for 24 h, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7.


HCT116 cells weretreated with AGK2 for 24 h ( g ) , and the cytosolic lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O

HCT116 cells weretreated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( a ) HCT116 cells or stable FoxO1 - RNAi HCT116 cells were treated with a non - specific RNAi control or SIRT2 - RNAi , and LC3 - II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A .

O O O B-CELL_LINE O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O

(a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A.


( b ) HCT116 cells or stable FoxO1 - RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h , in the presence or absence of E64 and pepstatin A . Changes in FoxO1 , p62 and LC3 - II were determined by western blotting ( upper panels ) , or the formation of LC3 punctate signals was observed ( lower panels ) . HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed . Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel . Data are means ± s . d . ( n = 3 ) .

O O O B-CELL_LINE O O O B-GENEPROD O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O

(b) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h, in the presence or absence of E64 and pepstatin A. Changes in FoxO1, p62 and LC3-II were determined by western blotting (upper panels), or the formation of LC3 punctate signals was observed (lower panels). HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed. Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel. Data are means ± s.d. (n = 3).


( c ) H1299 cells were transfected with Flag - FoxO1 ( WT ) , SIRT2 ( WT ) or SIRT2 ( 168A ) . At 24 h after transfection , the cell lysate was extracted and immunoblotted with anti - Flag , anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin . .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(c) H1299 cells were transfected with Flag-FoxO1(WT), SIRT2(WT) or SIRT2(168A). At 24 h after transfection, the cell lysate was extracted and immunoblotted with anti-Flag, anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin..


( d ) HCT116 cells were transfected with a control plasmid , a SIRT2 ( WT ) - expressing plasmid or a SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , the cells were subjected to serum starvation for 24 h . The cell lysate was extracted and immunoblotted with anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin .

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O 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-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O

(d) HCT116 cells were transfected with a control plasmid, a SIRT2(WT)-expressing plasmid or a SIRT2(168A)-expressing plasmid. At 24 h after transfection, the cells were subjected to serum starvation for 24 h. The cell lysate was extracted and immunoblotted with anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin.


( e ) H1299 cells were transfected with Myc - Atg7 , Flag - FoxO1 ( WT ) or mutated Flag - FoxO1 ( KR ) . At 24 h after transfection , the cytosolic lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O

(e) H1299 cells were transfected with Myc-Atg7, Flag-FoxO1(WT) or mutated Flag-FoxO1(KR). At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) H1299 cells were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( K262R ) , Flag - FoxO1 ( K265R ) , Flag - FoxO1 ( K274R ) or Flag - FoxO1 ( KR ) . At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted , the cell lysate was extracted and immunoblotted with anti - Flag , anti - LC3 , anti - p62 or anti - β - actin .

O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O

(f) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(K262R), Flag-FoxO1(K265R), Flag-FoxO1(K274R) or Flag-FoxO1(KR). At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted, the cell lysate was extracted and immunoblotted with anti-Flag, anti-LC3, anti-p62 or anti-β-actin.


( g ) Left : H1299 cells were co - transfected with GFP - LC3 , Flag - FoxO1 ( WT ) or Flag - FoxO1 ( KR ) . The formation of GFP - LC3 - labelled punctate signals was observed 24 h after transfection . Right : quantification of the ratio of GFP - LC3 - labelled punctate cells to overall survived H1299 cells . Data in g are means ± s . d . ( n = 3 ) . Scale bars , 25 μm ( b ) , 10 μm ( g ) .

O O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O 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) Left: H1299 cells were co-transfected with GFP-LC3, Flag-FoxO1(WT) or Flag-FoxO1(KR). The formation of GFP-LC3-labelled punctate signals was observed 24 h after transfection. Right: quantification of the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells. Data in g are means ± s.d. (n = 3). Scale bars, 25 μm (b), 10 μm (g).


( a ) H1299 cells were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( 3A ) , Flag - FoxO1 ( ΔDB ) or Flag - FoxO1 ( ΔDB3A ) , in the presence or absence of 10 mM 3 - MA ( upper panel ) or in the presence or absence of 50 μM Z - VAD - fmk ( lower panel ) ; cells were stained with propidium iodide ( PI ) 48 h after transfection . PI - stained ( dead ) cells were counted by flow cytometry and the percentage cell loss was calculated .

O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(a) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(3A), Flag-FoxO1(ΔDB) or Flag-FoxO1(ΔDB3A), in the presence or absence of 10 mM 3-MA (upper panel) or in the presence or absence of 50 μM Z-VAD-fmk (lower panel); cells were stained with propidium iodide (PI) 48 h after transfection. PI-stained (dead) cells were counted by flow cytometry and the percentage cell loss was calculated.


( b ) FoxO1 ( WT ) or Flag - FoxO1 ( ΔDB ) was transfected into H1299 cells ; 48 h later the autophagic vesicles were observed under an electron microscope . Enlarged images ( right ) show clearer autophagic vesicles indicated by arrows . Scale bars , 2 μm .

O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

(b) FoxO1(WT) or Flag-FoxO1(ΔDB) was transfected into H1299 cells; 48 h later the autophagic vesicles were observed under an electron microscope. Enlarged images (right) show clearer autophagic vesicles indicated by arrows. Scale bars, 2 μm.


( c ) 48 h cells with or without Atg5 RNAi were transfected with FoxO1 ( WT ) , and cell death was determined by staining with PI at 48 h after transfection . Data in c are means ± s . d . ( n = 3 ) .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(c) 48 h cells with or without Atg5 RNAi were transfected with FoxO1(WT), and cell death was determined by staining with PI at 48 h after transfection. Data in c are means ± s.d. (n = 3).


( d ) The stable Atg5 knockdown H1299 cells was further transfected with a control plasmid , Flag - FoxO1 ( WT ) , or Flag - FoxO1 ( ΔDB ) , and the percentage cell loss was quantified by flow cytometry at 48 h after transfection . Data in d are means ± s . d . ( n = 3 ) .

O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(d) The stable Atg5 knockdown H1299 cells was further transfected with a control plasmid, Flag-FoxO1(WT), or Flag-FoxO1(ΔDB), and the percentage cell loss was quantified by flow cytometry at 48 h after transfection. Data in d are means ± s.d. (n = 3).


( a ) Stable H1299 cells with or without Atg7 knockdown were transfected with a control plasmid ( group 1 ) , Flag - FoxO1 ( ΔDB ) ( group 2 ) or Flag - FoxO1 ( ΔDB - 3A ) ( group 3 ) ; 107 cells were then injected into 18 nude mice , with 6 mice per group . Seven weeks later the mice were killed and the tumours were weighed and analysed ( bottom graph ) . Group 1 : tumour weights were 1 . 22 ± 0 . 78 g ( without Atg7 RNAi ) and 1 . 31 ± 0 . 43 g ( with Atg7 RNAi ) , P = 0 . 426 . Group 2 : tumour weights were 0 . 49 ± 0 . 18 g ( without Atg7 RNAi ) and 0 . 80 ± 0 . 20 g ( with Atg7 RNAi ) , P = 0 . 017 . Group 3 : tumour weights were 1 . 00 ± 0 . 40 g ( without Atg7 RNAi ) and 1 . 10 ± 0 . 36 g ( with Atg7 RNAi ) , P = 0 . 500 . Data are means ± s . d . ( n = 6 ) .

O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O B-TISSUE O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(a) Stable H1299 cells with or without Atg7 knockdown were transfected with a control plasmid (group 1), Flag-FoxO1(ΔDB) (group 2) or Flag-FoxO1(ΔDB-3A) (group 3); 107 cells were then injected into 18 nude mice, with 6 mice per group. Seven weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Group 1: tumour weights were 1.22 ± 0.78 g (without Atg7 RNAi) and 1.31 ± 0.43 g (with Atg7 RNAi), P = 0.426. Group 2: tumour weights were 0.49 ± 0.18 g (without Atg7 RNAi) and 0.80 ± 0.20 g (with Atg7 RNAi), P = 0.017. Group 3: tumour weights were 1.00 ± 0.40 g (without Atg7 RNAi) and 1.10 ± 0.36 g (with Atg7 RNAi), P = 0.500. Data are means ± s.d. (n = 6).


( b ) Expression levels of FoxO1 and Atg7 conjugate in mice tumours were determined by western blotting .

O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY O

(b) Expression levels of FoxO1 and Atg7 conjugate in mice tumours were determined by western blotting.


( c ) HCT116 cells ( 107 ) or stable FoxO1 - knockdown HCT116 cells ( 107 ) were injected into the left or right hind legs of three nude mice . Six weeks later the mice were killed and the tumours were weighed and analysed ( bottom graph ) . Tumour weights were 0 . 08 ± 0 . 10 g ( without FoxO1 RNAi ) and 0 . 48 ± 0 . 06 g ( with FoxO1 RNAi ) , P = 0 . 020 . Data are means ± sd . n = 3 .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(c) HCT116 cells (107) or stable FoxO1-knockdown HCT116 cells (107) were injected into the left or right hind legs of three nude mice. Six weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Tumour weights were 0.08 ± 0.10 g (without FoxO1 RNAi) and 0.48 ± 0.06 g (with FoxO1 RNAi), P = 0.020. Data are means ± sd. n = 3.


( d ) Protein was extracted from one paired colon tumour ( T ) or adjacent normal tissue ( N ) from one patient , and western blotting was performed to detect changes in FoxO1 or p62 .

O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O

(d) Protein was extracted from one paired colon tumour (T) or adjacent normal tissue (N) from one patient, and western blotting was performed to detect changes in FoxO1 or p62.


( e ) Proteins were extracted from 16 paired samples from patients for western blotting with anti - FoxO1 or with anti - p62 antibody . Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β - actin band . FoxO1 of normal tissue , 0 . 37 ; FoxO1 of tumour tissue , 0 . 10 ; P = 0 . 001 , two - sided t - test . p62 of normal tissue , 0 . 32 ; p62 of tumour tissue , 1 . 00 ; P = 0 . 009 , two - sided t - test . Data in e are means ± s . d . ( n = 16 ) .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-DISEASE O O O O O O O O O O O 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) Proteins were extracted from 16 paired samples from patients for western blotting with anti-FoxO1 or with anti-p62 antibody. Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β-actin band. FoxO1 of normal tissue, 0.37; FoxO1 of tumour tissue, 0.10; P = 0.001, two-sided t-test. p62 of normal tissue, 0.32; p62 of tumour tissue, 1.00; P = 0.009, two-sided t-test. Data in e are means ± s.d. (n = 16).


Representative dual - channel kymographs showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative dual-channel kymographs showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Time - lapse images ( B ) showing colocalization and comigration ( white arrows ) of Rheb with mitochondria in a retrograde direction along axons during a 148 - s recording period . Cortical neurons were transfected with GFP - Rheb and DsRed - Mito at 5 - 7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10 - 12 DIV .

O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O

Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV.


quantitative analysis showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

O O O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

quantitative analysis showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . The percentages of mitochondria targeted by Rheb or Rheb - SSVM in the presence or absence of CCCP were quantified ,

O O O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O

Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively


representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . Rheb - SSVM , a farnesylation deficient mutant of Rheb , fails to be recruited to mitochondria upon dissipating ∆ ψm . Mitochondria associated with Rheb were marked by white arrows .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O

representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows.


F - H Representative dual - channel kymographs ( F ) and quantitative analysis ( G , H ) showing that Rheb - mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation . Note that Rheb , but not Rheb - SSVM , is associated with autophagic vacuoles ( AVs ) within axons upon ∆ ψm dissipation . Rheb - associated AVs move in a predominant retrograde direction along axons . The number of Rheb - associated AVs ( Rheb - AVs ) with or without CCCP treatment and the relative motilities of AVs and Rheb - AVs in CCCP - treated axons were quantified ,

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O

F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively


Representative dual - channel kymographs showing that Rheb enhances mitophagy in CCCP - treated axons .

O O O O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SUBCELLULAR O

Representative dual-channel kymographs showing that Rheb enhances mitophagy in CCCP-treated axons.


quantitative analysis showing that Rheb enhances mitophagy in CCCP - treated axons . The numbers of mitochondria ( Mito ) , AVs , and Mito - AVs / mitophagosomes , the percentage of Mito within AVs , were

O O O O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O

quantitative analysis showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, were quantified


quantitative analysis the percentage of Mito within AVs , and the motilities of AVs and Mito - AVs along axons with and without elevated Rheb expression were

O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O O

quantitative analysis the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantified


D , E Representative images ( D ) and quantitative analysis ( E ) showing that mitophagy in axons requires Rheb and Nix , but not Parkin . The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP - treated axons expressing scrambled shRNA , Rheb shRNA , Nix shRNA , or Parkin shRNA were quantified , respectively ( E ) .

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D,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E).


F , G Quantitative analysis ( F ) and representative blots ( G ) showing increased mitochondrial association with Rheb and LC3 - II in mature cortical neurons upon ∆ ψm depolarization . Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post - nuclear supernatant ( P ) , mitochondria - enriched membrane fraction ( M ) , and cytosolic supernatant ( S ) . Equal amounts of protein ( 20 μg ) were sequentially immunoblotted with antibodies against mitophagic / autophagic proteins Rheb , LC3 , and Rab7 , mitochondrial protein VDAC , and cytosolic protein GAPDH on the same membranes after stripping between each antibody application . The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH , compared to post - nuclear supernatant . Protein levels in mitochondrial fractions of CCCP - treated neurons were normalized to those in neurons incubated with DMSO . Data were quantified from four independent repeats .

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F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats.


Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP - treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA ( H ) .

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Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H).