Peak amplitudes of Ca2 + release in cells of the indicated genotypes in response to GPN ( F ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Peak amplitudes of Ca2+ release in cells of the indicated genotypes in response to GPN (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Ratio of the amplitudes of Ca2 + release in cells of the indicated genotypes in response to ML - SA1 and GPN . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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

Ratio of the amplitudes of Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Total Ca2 + release in cells of the indicated genotypes in response to ML - SA1 and GPN . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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

Total Ca2+ release in cells of the indicated genotypes in response to ML-SA1 and GPN. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Bar graphs showing the relative cell numbers in the indicated cell types following the indicated treatments . Cell numbers were assessed after a 48 - hour period of growth in cultures seeded at equal densities . All values represent mean ± SEM . Data points represent values from biological replicates .

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Bar graphs showing the relative cell numbers in the indicated cell types following the indicated treatments. Cell numbers were assessed after a 48-hour period of growth in cultures seeded at equal densities. All values represent mean±SEM. Data points represent values from biological replicates.


Representative images showing Ki67 - labeling in xenografts generated from implantation of HN31 cells stably expressing control shRNA ( left ) or MCOLN1 shRNA ( right ) . Pseudocolored panels show the representative distribution Ki67 positive or negative nuclei . Scale bars shown ( 0 . 5 mm ) apply to all panels .

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Representative images showing Ki67-labeling in xenografts generated from implantation of HN31 cells stably expressing control shRNA (left) or MCOLN1 shRNA (right). Pseudocolored panels show the representative distribution Ki67 positive or negative nuclei. Scale bars shown (0.5 mm) apply to all panels.


Bar graphs showing fraction ( left ) and total number ( right ) of Ki67 labeled nuclei in the indicated cell types . All values represent mean ± SEM . Values in the bar graph on the right were normalized to control mean . Data points represent values from independent eggs . Statistical test employed was Student ' s t - test .

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Bar graphs showing fraction (left) and total number (right) of Ki67 labeled nuclei in the indicated cell types. All values represent mean±SEM. Values in the bar graph on the right were normalized to control mean. Data points represent values from independent eggs. Statistical test employed was Student's t-test.


Bar graphs showing the relative cell numbers in UMSCC - 22A - HRASWT cells following the indicated perturbations . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graphs showing the relative cell numbers in UMSCC-22A-HRASWT cells following the indicated perturbations. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Kaplan - Meier curves showing the survival of patients stratified on the basis of MCOLN1 expression in BLCA and HNSC patients whose tumors were wild - type for HRAS ( right ) or carried oncogenic HRAS mutations ( left ) . Statistical test employed was the Mantel - Cox log - rank test .

Kaplan-Meier curves showing the survival of patients stratified on the basis of MCOLN1 expression in BLCA and HNSC patients whose tumors were wild-type for HRAS (right) or carried oncogenic HRAS mutations (left). Statistical test employed was the Mantel-Cox log-rank test.


Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML - SI1 as indicated . The primary antibodies used are indicated on the right . Bar graph showing quantification of the Western blots shown in ( A ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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

Representative Western blots generated using extracts from cells of expressing oncogenic HRAS and controls treated with ML-SI1 as indicated. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (A). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations . Primary antibodies used are indicated on the right .

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Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Primary antibodies used are indicated on the right.


Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations . Bar graph showing quantification of the Western blots in ( C ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (C). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations . Bar graph showing quantification of the Western blots in ( D ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

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Representative Western blots generated using extracts from the indicated cell types after the indicated perturbations. Bar graph showing quantification of the Western blots in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing relative levels of trpml mRNA in Drosophila larval brain extracts from control animals and animals expressing dRasG12V in glia . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing relative levels of trpml mRNA in Drosophila larval brain extracts from control animals and animals expressing dRasG12V in glia. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative images of Drosophila 3rd instar larvae expressing the indicated transgenes in glial cells . repo - GAL4 was used to drive the expression of UAS - dRasG12V in glia . Images shown are volumetric reconstructions of confocal stacks . The various anatomical structures of the larval brain are annotated . VNC refers to the ' ventral nerve chord ' , which is distinct from dorsal hemispheres of the brain . GFP staining represents the total glial membranes in the larval brains . Scale bar , 100μm .

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Representative images of Drosophila 3rd instar larvae expressing the indicated transgenes in glial cells. repo-GAL4 was used to drive the expression of UAS-dRasG12V in glia. Images shown are volumetric reconstructions of confocal stacks. The various anatomical structures of the larval brain are annotated. VNC refers to the 'ventral nerve chord', which is distinct from dorsal hemispheres of the brain. GFP staining represents the total glial membranes in the larval brains. Scale bar, 100μm.


Bar graph showing relative glial cell volume in larvae of the indicated genotypes . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing relative glial cell volume in larvae of the indicated genotypes. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of the indicated genotypes . He - GAL4 was used to drive the expression of UAS - dRasG12V in macrophages . The primary antibodies used are indicated on the right . Bar graph showing quantification of the Western blots shown in ( D ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Representative Western blots generated using macrophage extracts isolated from 3rd instar larvae of the indicated genotypes. He-GAL4 was used to drive the expression of UAS-dRasG12V in macrophages. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (D). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Representative Western blots generated using fat - body extracts isolated from 3rd instar larvae of the indicated genotypes . cg - GAL4 was used to drive the expression of UAS - HRASG12V in larval fat - bodies . The primary antibodies used are indicated on the right . Bar graph showing quantification of the Western blots shown in ( F ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

Representative Western blots generated using fat-body extracts isolated from 3rd instar larvae of the indicated genotypes. cg-GAL4 was used to drive the expression of UAS-HRASG12V in larval fat-bodies. The primary antibodies used are indicated on the right. Bar graph showing quantification of the Western blots shown in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Weighted mean univariate K - function curves of immunogold labeled GFP - HRASG12V under indicated conditions , n ≥ 15 for each condition . The gold point patterns were analyzed with univariate K - functions expressed as L ( r ) - r . The bar graph on the right shows the peak value Lmax of the L ( r ) - r curve , which represents the extent of GFP - HRASG12V nanoclustering . Values are normalized to DMSO average and represent mean ± SEM ( n ≥ 15 for each condition ) . Statistical significances of differences were evaluated in bootstrap tests .

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Weighted mean univariate K-function curves of immunogold labeled GFP-HRASG12V under indicated conditions, n ≥ 15 for each condition. The gold point patterns were analyzed with univariate K-functions expressed as L(r)-r. The bar graph on the right shows the peak value Lmax of the L(r)-r curve, which represents the extent of GFP-HRASG12V nanoclustering. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.


Bar graph showing GFP - HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs . Values are normalized to DMSO average and represent mean ± SEM , n ≥ 15 for each condition . Statistical test employed was Student ' s t - test with Bonferroni post hoc correction to account for multiple pairwise comparisons .

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Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. Values are normalized to DMSO average and represent mean±SEM, n ≥ 15 for each condition. Statistical test employed was Student's t-test with Bonferroni post hoc correction to account for multiple pairwise comparisons.


The extent of GFP - HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations . Values are normalized to DMSO average and represent mean ± SEM ( n ≥ 15 for each condition ) . Statistical significances of differences were evaluated in bootstrap tests .

The extent of GFP-HRASG12V nanoclustering quantified by Lmax values in cells exposed to the indicated perturbations. Values are normalized to DMSO average and represent mean±SEM (n ≥ 15 for each condition). Statistical significances of differences were evaluated in bootstrap tests.


Bar graph showing GFP - HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs . but in cells exposed to the indicated perturbations . Values shown are normalized to DMSO average .

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Bar graph showing GFP-HRASG12V content in the inner leaflet of the plasma membrane is quantified as the number of gold particles per 1μm2 region from electron micrographs. but in cells exposed to the indicated perturbations. Values shown are normalized to DMSO average.


Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA . Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom . Primary antibodies used are indicated on the right .

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

Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Primary antibodies used are indicated on the right.


Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA . Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom . Bar graph showing quantification of the Western blots in ( F ) . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was ANOVA .

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

Representative Western blots generated using extracts from HN31 cells stably expressing the MCOLN1 shRNA. Samples treated with exogenous cholesterol for 3 hours are indicated at the bottom. Bar graph showing quantification of the Western blots in (F). All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was ANOVA.


Bar graph showing the relative levels of total in HN31 cell extracts exposed to the indicated perturbagens Values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

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Bar graph showing the relative levels of total in HN31 cell extracts exposed to the indicated perturbagens Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing the relative levels of free / total cholesterol in HN31 cell extracts exposed to the indicated perturbagens Values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

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Bar graph showing the relative levels of free/total cholesterol in HN31 cell extracts exposed to the indicated perturbagens Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Bar graph showing the relative levels of free / total cholesterol in larval brain extracts from animals of the indicated genotypes Values represent mean ± SEM . Data points represent values from biological replicates . Statistical tests employed were Student ' s t - test and ANOVA .

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

Bar graph showing the relative levels of free/total cholesterol in larval brain extracts from animals of the indicated genotypes Values represent mean±SEM. Data points represent values from biological replicates. Statistical tests employed were Student's t-test and ANOVA.


Confocal images of MDCK cells expressing mCherry - D4H . Colocalization with the cortical actin ( phalloidin ) indicates that mCherry - D4H is predominantly localized to the plasma membrane in DMSO treated cells . Within 24 hours of ML - SI1 treatment , the mCherry - D4H redistributes to intracellular punctae . Although minimal mCherry - D4H signal remained at the plasma membrane in ML - SI1 - treated cells , the phalloidin signal ( cortical actin ) remained unchanged . Scale bar ( 10μm ) shown in the panel on the top right applies to all panels . Bar graph showing quantification of mCherry - D4H punctae in DMSO or ML - SI1 - treated cells . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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

Confocal images of MDCK cells expressing mCherry-D4H. Colocalization with the cortical actin (phalloidin) indicates that mCherry-D4H is predominantly localized to the plasma membrane in DMSO treated cells. Within 24 hours of ML-SI1 treatment, the mCherry-D4H redistributes to intracellular punctae. Although minimal mCherry-D4H signal remained at the plasma membrane in ML-SI1-treated cells, the phalloidin signal (cortical actin) remained unchanged. Scale bar (10μm) shown in the panel on the top right applies to all panels. Bar graph showing quantification of mCherry-D4H punctae in DMSO or ML-SI1-treated cells. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Confocal images of MDCK cells expressing mCherry - D4H and LAMP1 - GFP treated with ML - SI1 . Scale bar ( 10μm ) shown in the panel on the top right applies to all panels .

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Confocal images of MDCK cells expressing mCherry-D4H and LAMP1-GFP treated with ML-SI1. Scale bar (10μm) shown in the panel on the top right applies to all panels.


Confocal images of control and MCOLN1 shRNA - expressing HN31 cells treated with the cholesterol sensor , filipin . Scale bar ( 10μm ) shown in the panel on the top applies to both panels . Bar graph showing the quantification of the internalized filipin staining in HN31 cells after MCOLN1 knockdown . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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

Confocal images of control and MCOLN1 shRNA-expressing HN31 cells treated with the cholesterol sensor, filipin. Scale bar (10μm) shown in the panel on the top applies to both panels. Bar graph showing the quantification of the internalized filipin staining in HN31 cells after MCOLN1 knockdown. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Fraction of phase - separated GPMVs at the indicated temperatures . Colors of the lines indicate the treatment conditions of MDCK cells prior to generation of GPMVs . Dashed vertical lines indicate the miscibility temperature of the indicated populations of GPMVs . Values shown represent mean ± SEM . Lines were generated by fitting the data to sigmoidal functions .

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Fraction of phase-separated GPMVs at the indicated temperatures. Colors of the lines indicate the treatment conditions of MDCK cells prior to generation of GPMVs. Dashed vertical lines indicate the miscibility temperature of the indicated populations of GPMVs. Values shown represent mean±SEM. Lines were generated by fitting the data to sigmoidal functions.


Bar graph showing miscibility temperatures of GPMVs isolated from MDCK cells exposed to the indicated conditions . All values represent mean ± SEM . Data points represent values from biological replicates . Statistical test employed was Student ' s t - test .

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Bar graph showing miscibility temperatures of GPMVs isolated from MDCK cells exposed to the indicated conditions. All values represent mean±SEM. Data points represent values from biological replicates. Statistical test employed was Student's t-test.


Quantitative expression of Lif mRNA in the colon of DSS - challenged and control mice ( n = 6 per group ) .

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Quantitative expression of Lif mRNA in the colon of DSS-challenged and control mice (n=6 per group).


LIF measured by ELISA in mouse colon explant supernatants from DSS - challenged and control mice ( n = 4 per group ) .

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LIF measured by ELISA in mouse colon explant supernatants from DSS-challenged and control mice (n=4 per group).


Quantitative expression of Lif mRNA in LPLs and IECs from DSS - challenged and control mice ( n = 4 per group ) .

Quantitative expression of Lif mRNA in LPLs and IECs from DSS-challenged and control mice (n=4 per group).


Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics ( Abx ) at the indicated time points during colitis induction ( n = 3 per group ) .

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Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics (Abx) at the indicated time points during colitis induction (n=3 per group).


Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction ( n = 3 per group ) .

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Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction (n=3 per group).


mRNA expression of the indicated genes in IECs stimulated by LPS ( 2 . 5μg / ml ) , PGN ( 3μg / ml ) or LTA ( 2μg / ml ) for 2 hours .

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mRNA expression of the indicated genes in IECs stimulated by LPS (2.5μg/ml), PGN (3μg/ml) or LTA (2μg/ml) for 2 hours.


Serum concentrations of LIF in IEC supernatants untreated ( G ) followed by LPS ( 2 . 5μg / ml ) or LTA ( 2μg / ml ) for 12hours , as determined by ELISA .

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Serum concentrations of LIF in IEC supernatants untreated (G) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.


Serum concentrations of LIF in IEC supernatants pretreated with monensin ( H ) followed by LPS ( 2 . 5μg / ml ) or LTA ( 2μg / ml ) for 12hours , as determined by ELISA .

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Serum concentrations of LIF in IEC supernatants pretreated with monensin (H) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA.


Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4 + T cells ( I ) and IECs ( J ) treated with LIF ( 20 ng / ml ) at different time points . The lower panel shows the intensity analysis of the bands from three independent experiments .

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Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4+ T cells (I) and IECs (J) treated with LIF (20 ng/ml) at different time points. The lower panel shows the intensity analysis of the bands from three independent experiments.


Stat4 mRNA expression in LPLs and IECs ( n = 6 ) .

Stat4 mRNA expression in LPLs and IECs (n=6).


The proliferation rate of CD4 + T cells ( L ) and DLD - 1 cells ( M ) was determined in the presence or absence of LIF .

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The proliferation rate of CD4+ T cells (L) and DLD-1 cells (M) was determined in the presence or absence of LIF.


Weight loss of wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS .

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Weight loss of wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS.


wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . Macroscopic changes in the colon of the mice on day 10 of colitis induction Comparison of the colon length in the mice on day 10 of colitis

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wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. Macroscopic changes in the colon of the mice on day 10 of colitis induction Comparison of the colon length in the mice on day 10 of colitis induction


wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . DAI ( stool consistency and bleeding score ) of colitis mice

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wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. DAI (stool consistency and bleeding score) of colitis mice treated


wild - type ( WT ) ( n = 6 ) or Stat4 knockout ( Stat4 - KO ) ( n = 6 ) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3 % DSS . H & E histology of representative colons from colitis mice . Scale bar , 20μm .

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wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS. H&E histology of representative colons from colitis mice. Scale bar, 20μm.


Weight loss of Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks .

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Weight loss of Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks.


Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks . Macroscopic changes in the colon of Rag1 - / - mice treated Comparison of colon lengths in the Rag1 - / - mice at week 8 ( n = 5 ) .

Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. Macroscopic changes in the colon of Rag1-/- mice treated Comparison of colon lengths in the Rag1-/- mice at week 8 (n=5).


Rag1 - / - ( n = 6 ) mice transferred with CD45RBhi CD4 + T cells and intraperitoneally injected with PBS or LIF for 8 weeks . H & E histology of representative mouse colons on day 54 of colitis

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

Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks. H&E histology of representative mouse colons on day 54 of colitis induction


Colons obtained from WT or Stat4 - KO mice on day10 of the colitis model induced as in Figure 2A were immunostained with an antibody against phosphorylated STAT4 . Scale bar , 50μm .

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

Colons obtained from WT or Stat4-KO mice on day10 of the colitis model induced as in Figure 2A were immunostained with an antibody against phosphorylated STAT4. Scale bar, 50μm.


Immunoblot analysis of STAT4 phosphorylation in spleen tissue from colitis mice .

Immunoblot analysis of STAT4 phosphorylation in spleen tissue from colitis mice.


FACS staining of LPLs isolated on day10 from the colon of WT or Stat4 - KO colitis mice receiving PBS or LIF ( n = 4 per group ) . The percentage of IL - 17A + and / or IFNγ + CD4 + T cells in vivo was analyzed .

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

FACS staining of LPLs isolated on day10 from the colon of WT or Stat4-KO colitis mice receiving PBS or LIF (n=4 per group). The percentage of IL-17A+ and/or IFNγ+ CD4+ T cells in vivo was analyzed.


Quantitative expression of Il17a mRNA in MLNs and LNs from colitis mice treated


FACS staining of WT and Stat4 - KO naïve CD4 + T cells treated with LIF ( 50 ng / ml ) on day 4 of induction into Th17 cell subsets .

FACS staining of WT and Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into Th17 cell subsets.


qPCR analysis of Th17 - related genes in WT or Stat4 - KO naïve CD4 + T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF ( n = 3 per group ) .

qPCR analysis of Th17-related genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF (n=3 per group).


FACS staining of WT or Stat4 - KO naïve CD4 + T cells treated with LIF ( 50 ng / ml ) on day 4 of induction into nonpathogenic or pathogenic Th17 cells .

FACS staining of WT or Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into nonpathogenic or pathogenic Th17 cells.


Immunoblot analysis of STAT4 phosphorylation in CD4 + T cells pretreated with PBS or LY2784544 ( 1μM ) for 12 hours followed by LIF for the indicated number of minutes .

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

Immunoblot analysis of STAT4 phosphorylation in CD4+ T cells pretreated with PBS or LY2784544 (1μM) for 12 hours followed by LIF for the indicated number of minutes.


Immunoblot analysis of CD4 + T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti - LIFR antibody .

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

Immunoblot analysis of CD4+ T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti-LIFR antibody.


Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs . The red asterisks denote phosphorylated STAT bands .

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

Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs. The red asterisks denote phosphorylated STAT bands.


The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus .

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

The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus.


Immunoblot analysis of the indicated protein modifications in the total lysate of CD4 + T cells treated with LIF ( 20 ng / ml ) at different time points .

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

Immunoblot analysis of the indicated protein modifications in the total lysate of CD4+ T cells treated with LIF (20 ng/ml) at different time points.


Coimmunoprecipitation and immunoblot analysis of LIF - treated HEK293T cells cotransfected with the indicated constructs .

Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs.


Immunoblot analysis of the distribution of the indicated proteins in the CD4 + T cell fraction .

Immunoblot analysis of the distribution of the indicated proteins in the CD4+ T cell fraction.


Cultured HeLa cells transfected with GFP - tagged STAT constructs were exposed to LIF and imaged 30 minutes later . Scale bar , 50μm .

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

Cultured HeLa cells transfected with GFP-tagged STAT constructs were exposed to LIF and imaged 30 minutes later. Scale bar, 50μm.


Heatmap showing Th17 signature genes ( left ) or Th17 regulatory genes ( right ) up - or downregulated in WT or Stat4 - KO LPLs by LIF treatment ( n = 2 ) .

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

Heatmap showing Th17 signature genes (left) or Th17 regulatory genes (right) up- or downregulated in WT or Stat4-KO LPLs by LIF treatment (n=2).


Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice ( n = 3 per group ) .

Quantitative mRNA expression analysis of the indicated genes in LPLs from colitis mice (n=3 per group).


Quantitative mRNA expression analysis of the indicated genes in WT or Stat4 - KO naïve CD4 + T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF .

O O O O O O O O O O O O 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 mRNA expression analysis of the indicated genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF.


The GAS - luciferase reporter activity assay was performed in Stat3 - / - MEFs transfected with empty vector ( EV ) or STAT4 constructs and then treated with LIF for 8 hours .

The GAS-luciferase reporter activity assay was performed in Stat3-/- MEFs transfected with empty vector (EV) or STAT4 constructs and then treated with LIF for 8 hours.


Relative SIE - ( I ) activities in Stat3 - / - MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours .

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

Relative SIE-(I) activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours.


Relative AGG - luciferase reporter activities in Stat3 - / - MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours .

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

Relative AGG-luciferase reporter activities in Stat3-/- MEFs transfected with EV or the indicated STAT constructs and treated with LIF for 8 hours.


ChIP analysis of naïve CD4 + T cells obtained from IL - 17A - eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days . GFP + cells isolated by flow cytometry were restimulated with IL - 6 in the presence or absence of LIF , crosslinked with formaldehyde and immunoprecipitated with anti - STAT3 ( top ) or anti - STAT4 ( bottom ) antibodies , followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1 - p10 primer pairs . The results are presented relative to the amount of input DNA .

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

ChIP analysis of naïve CD4+ T cells obtained from IL-17A-eGFP mice cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. GFP+ cells isolated by flow cytometry were restimulated with IL-6 in the presence or absence of LIF, crosslinked with formaldehyde and immunoprecipitated with anti-STAT3 (top) or anti-STAT4 (bottom) antibodies, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.


ChIP analysis of naïve WT or Stat4 - KO CD4 + T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days . The cells were restimulated with IL - 6 or LIF for 30 minutes , crosslinked with formaldehyde and immunoprecipitated with the anti - STAT3 antibody , followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1 - p10 primer pairs . The results are presented relative to the amount of input DNA .

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

ChIP analysis of naïve WT or Stat4-KO CD4+ T cells cultured under Th0 or Th17 conditions in the absence or presence of LIF for 3 days. The cells were restimulated with IL-6 or LIF for 30 minutes, crosslinked with formaldehyde and immunoprecipitated with the anti-STAT3 antibody, followed by the amplification of the immunoprecipitated DNA by quantitative PCR with the p1-p10 primer pairs. The results are presented relative to the amount of input DNA.


Relative Il17a - SIE - ( left ) or AGG - luciferase reporter ( right ) activities in Stat3 - / - MEFs transfected with EV , STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL - 6 or LIF for 8 hours .

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

Relative Il17a-SIE- (left) or AGG-luciferase reporter (right) activities in Stat3-/- MEFs transfected with EV, STAT3 or STAT4 or cotransfected with STAT3 and STAT4 and then treated with IL-6 or LIF for 8 hours.


Relative Il17a promoter - luciferase reporter activities in Stat3 - / - MEFs transfected with the indicated constructs and then treated with LIF for 8 hours .

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

Relative Il17a promoter-luciferase reporter activities in Stat3-/- MEFs transfected with the indicated constructs and then treated with LIF for 8 hours.


Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A . The number of proliferating cells per crypt in ( A ) ( upper panel ) or apoptotic cells per field in ( B ) was determined ( n = 5 per group , 5 crypts were counted per mouse ) . Scale bar , 50μm .

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

Ki67 and cleaved caspase3 staining of representative colons obtained from mice on day10 of colitis induction as described in Figure 2A. The number of proliferating cells per crypt in (A) (upper panel) or apoptotic cells per field in (B) was determined (n=5 per group, 5 crypts were counted per mouse). Scale bar, 50μm.


Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4 - KO colitis mice

Quantitative mRNA expression analysis of the indicated genes in colon tissues obtained from WT or Stat4-KO colitis mice treated


Immunoblot analysis of the expression and modification of the indicated proteins in DLD - 1 cells treated with LIF ( 20 ng / ml ) for different durations .

Immunoblot analysis of the expression and modification of the indicated proteins in DLD-1 cells treated with LIF (20 ng/ml) for different durations.


Immunoblot analysis of YAP expression in WT or Stat4 - KO IECs treated with LIF ( 20 ng / ml ) for different durations ; β - actin was used as a loading control ( top ) . The statistical analysis of YAP protein intensity is shown in the bottom panel .

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

Immunoblot analysis of YAP expression in WT or Stat4-KO IECs treated with LIF (20 ng/ml) for different durations; β-actin was used as a loading control (top). The statistical analysis of YAP protein intensity is shown in the bottom panel.


Relative YAP promoter - luciferase reporter activities in HEK293T cells transfected with EV , STAT3 or STAT4 and then treated with IL - 6 or LIF for 8 hours .

Relative YAP promoter-luciferase reporter activities in HEK293T cells transfected with EV, STAT3 or STAT4 and then treated with IL-6 or LIF for 8 hours.


Heatmap showing the expression profile of cell proliferation - related genes in LIF - treated or control IECs ( Ctrl ) ( K ) .

Heatmap showing the expression profile of cell proliferation-related genes in LIF-treated or control IECs (Ctrl) (K).


Principal coordinate analysis ( PCoA ) of the microbiota composition determined by 16S rRNA gene sequencing of day 0 and day 9 fecal specimens from mice treated Each symbol represents an individual mouse . PC1 and PC2 represent principal components 1 and 2 , respectively .

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

Principal coordinate analysis (PCoA) of the microbiota composition determined by 16S rRNA gene sequencing of day 0 and day 9 fecal specimens from mice treated Each symbol represents an individual mouse. PC1 and PC2 represent principal components 1 and 2, respectively.


The bars depict the average relative abundance of the bacterial phyla of the microbiota in fecal specimens , as determined by taxon - based analyses as described in ( A ) ( n = 5 per group ) .

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

The bars depict the average relative abundance of the bacterial phyla of the microbiota in fecal specimens, as determined by taxon-based analyses as described in (A) (n=5 per group).


16S rRNA sequencing analyses of the Proteobacteria distribution in feces from WT or Stat4 - KO colitis mice . ( n = 5 per group ) .

16S rRNA sequencing analyses of the Proteobacteria distribution in feces from WT or Stat4-KO colitis mice. (n=5 per group).


Heatmap depicting the relative abundance of microbes at the family level ( top ) and the genus level ( bottom ) in the fecal specimens described in ( A ) ( n = 3 per group ) .

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

Heatmap depicting the relative abundance of microbes at the family level (top) and the genus level (bottom) in the fecal specimens described in (A) (n=3 per group).


qPCR of the 16S rRNA genes of microbes in the phylum Proteobacteria , family Enterobacteriaceae , and genera Escherichia - Shigella in the feces of the indicated mice on day0 and day10 ( n = 6 per group ) of colitis induction .

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

qPCR of the 16S rRNA genes of microbes in the phylum Proteobacteria, family Enterobacteriaceae, and genera Escherichia-Shigella in the feces of the indicated mice on day0 and day10 (n=6 per group) of colitis induction.


Quantitative mRNA expression analysis of proinflammatory genes in the colon of mice ( n = 3 per group )

Quantitative mRNA expression analysis of proinflammatory genes in the colon of mice (n=3 per group) treated


The MR tail is the main contributor for Nek2A ubiquitination . Ubiquitination reactions performed with APC / CCdc20 for Nek2A wild type ( Nek2AWT ) and degron mutants . Nek2AK is the KEN box mutant ( 391KEN393 / KAA ) , Nek2AK / D is the ( KEN and D box mutant ( 391KEN393 / KAA , 361RKFL364 / AKFA ) , Nek2AMR is the 443 ∆ MR mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 MR tail is the main contributor for Nek2A ubiquitination. Ubiquitination reactions performed with APC/CCdc20 for Nek2A wild type (Nek2AWT) and degron mutants. Nek2AK is the KEN box mutant (391KEN393/KAA), Nek2AK/D is the (KEN and D box mutant (391KEN393/KAA, 361RKFL364/AKFA), Nek2AMR is the 443∆MR mutant.


The APC / C requires the Cdc20 coactivator subunit to ubiquitinate Nek2A . Nek2A ubiquitination reactions performed with the APC / C in absence and in presence of increasing concentrations of Cdc20 using the E2 UbcH10 .

O O O O O O O O O O O O O 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 APC/C requires the Cdc20 coactivator subunit to ubiquitinate Nek2A. Nek2A ubiquitination reactions performed with the APC/C in absence and in presence of increasing concentrations of Cdc20 using the E2 UbcH10.


The MR tail is essential for Nek2A binding to the APC / C whereas in contrast Cdc20 is dispensable . Size exclusion chromatography peak fractions of APC / C complexes with Nek2A . The input material ( 13 % of total ) is indicated ( i ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 MR tail is essential for Nek2A binding to the APC/C whereas in contrast Cdc20 is dispensable. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A. The input material (13% of total) is indicated (i).


Nek2A binding does not stabilize binding of the coactivator subunit Cdc20 to the APC / C and does not compete with cyclin A for binding to the APC / C . Size exclusion chromatography peak fractions of APC / C complexes with Nek2A .

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

Nek2A binding does not stabilize binding of the coactivator subunit Cdc20 to the APC/C and does not compete with cyclin A for binding to the APC/C. Size exclusion chromatography peak fractions of APC/C complexes with Nek2A.


Nek2A ubiquitination reactions performed with either APC / CCdc20 wild type ( APC / CWT ) or mutants . APC / C2m is the N392A / E395A mutant of Apc2 . APC / C2 / 4m is the N392A / E395A / R48A / H53A / S51A mutant of Apc2 and Apc4 .

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

Nek2A ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants. APC/C2m is the N392A/E395A mutant of Apc2. APC/C2/4m is the N392A/E395A/R48A/H53A/S51A mutant of Apc2 and Apc4.


Securin ubiquitination reactions performed with either APC / CCdc20 wild type ( APC / CWT ) or mutants .

Securin ubiquitination reactions performed with either APC/CCdc20 wild type (APC/CWT) or mutants.


Ubiquitination reactions of either Nek2A or securin by the APC / CCdc20 and the effect of increasing concentrations of the MCC .

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

Ubiquitination reactions of either Nek2A or securin by the APC/CCdc20 and the effect of increasing concentrations of the MCC.


Size exclusion chromatography peak fractions of APC / C complexes with either

Size exclusion chromatography peak fractions of APC/C complexes with either Nek2A


Size exclusion chromatography peak fractions of APC / C complexes with Nek2A APC / CΔApc15 is the Apc15 deleted mutant APC / C ,

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

Size exclusion chromatography peak fractions of APC/C complexes with Nek2A APC/CΔApc15 is the Apc15 deleted mutant APC/C,


Size exclusion chromatography peak fractions of APC / C complexes with Nek2A MCCΔIR is the Cdc20 IR tail deleted mutant MCC . MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1

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

Size exclusion chromatography peak fractions of APC/C complexes with Nek2A MCCΔIR is the Cdc20 IR tail deleted mutant MCC. MCC-BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1


Size exclusion chromatography peak fractions of APC / C complexes with Nek2A MCCΔIR is the Cdc20 IR tail deleted mutant MCC . MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on

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


Size exclusion chromatography peak fractions of APC / C complexes securin MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1

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

Size exclusion chromatography peak fractions of APC/C complexes securin MCC-BubR1Wm is the MCC mutant at the APC2WHB binding surface on BubR1


Size exclusion chromatography peak fractions of APC / C complexes securin MCC - BubR1Wm is the MCC mutant at the APC2WHB binding surface on


Ubiquitination reactions of cyclin A by the APC / CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5 .

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

Ubiquitination reactions of cyclin A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.


Ubiquitination reactions of Nek2A by the APC / CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5 .

Ubiquitination reactions of Nek2A by the APC/CCdc20 in presence of increasing concentrations of either UbcH10 or UbcH5.


Western blot of Hek293 cells showing the siRNA - mediated depletion of UbcH5 , UbcH10 , or both E2 enzymes together . Tubulin serves a loading control .

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

Western blot of Hek293 cells showing the siRNA-mediated depletion of UbcH5, UbcH10, or both E2 enzymes together. Tubulin serves a loading control.


Exemplary still images from time courses between NEBD and anaphase of eGFP - Nek2A degradation in HEK cells . Cells were either treated with siGL2 as control or depleted of the indicated E2 enzymes . The chromosomes are coloured in cyan and eGFP - Nek2A in green , with the outline of the cells are indicated with dashed yellow lines . Time is given as hh : mm . Scale bar 10 μm .

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

Exemplary still images from time courses between NEBD and anaphase of eGFP-Nek2A degradation in HEK cells. Cells were either treated with siGL2 as control or depleted of the indicated E2 enzymes. The chromosomes are coloured in cyan and eGFP-Nek2A in green, with the outline of the cells are indicated with dashed yellow lines. Time is given as hh:mm. Scale bar 10 μm.


Degradation profiles of eGFP - cyclin A2 ( top ) and eGFP - Nek2A ( bottom ) in HEK cells during mitosis . The timepoint of NEBD is marked at 0 minutes in the graphs . Asterisks indicate values that are significantly different from the same time point of the siGL2 control as determined by a Mann - Whitney U - test Mean ± s . d . is shown . The number of cells analysed are N = 66 ( Cyclin A2 siGL2 ) , 52 ( Cyclin A2 siUbcH5 ) , 28 ( Cyclin A2 siUbcH10 ) , 27 ( Cyclin A2 siU5 / siU10 ) , 80 ( Nek2A siGL2 ) , 64 ( Nek2A siUbcH5 ) , 21 ( Nek2A siUbcH10 ) , 32 ( Nek2A siU5 / siU10 ) . All data are from at least two biological replicates .

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

Degradation profiles of eGFP-cyclin A2 (top) and eGFP-Nek2A (bottom) in HEK cells during mitosis. The timepoint of NEBD is marked at 0 minutes in the graphs. Asterisks indicate values that are significantly different from the same time point of the siGL2 control as determined by a Mann-Whitney U-test Mean ± s.d. is shown. The number of cells analysed are N = 66 (Cyclin A2 siGL2), 52 (Cyclin A2 siUbcH5), 28 (Cyclin A2 siUbcH10), 27 (Cyclin A2 siU5/siU10), 80 (Nek2A siGL2), 64 (Nek2A siUbcH5), 21 (Nek2A siUbcH10), 32 (Nek2A siU5/siU10). All data are from at least two biological replicates.


( a , b ) HeLa cells were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( a ) Endogenous LC3 was detected by immunoblot in total cell lysates , quantified by densitometric analysis and normalized to actin . Relative levels were expressed in percentage with control cells set to 100 and shown in graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h. (a) Endogenous LC3 was detected by immunoblot in total cell lysates, quantified by densitometric analysis and normalized to actin. Relative levels were expressed in percentage with control cells set to 100 and shown in graph.


( a , b ) HeLa cells were treated with 10 μg ml − 1 N - Shh peptide for 24 h . ( b ) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels . Graph shows mRNA levels quantified by densitometric analysis and normalized to actin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,b) HeLa cells were treated with 10 μg ml−1 N-Shh peptide for 24 h.(b) mRNA was obtained and levels of Ptch1 or Gli1 and actin mRNA were amplified by standard PCR and visualized in agarose gels. Graph shows mRNA levels quantified by densitometric analysis and normalized to actin.


( c ) 8xGli - luciferase construct was transfected in HeLa cells for 24 h , followed by treatment with 10 μM purmorphamine , or DMSO as a control , for another 24 h . Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control .

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

(c) 8xGli-luciferase construct was transfected in HeLa cells for 24 h, followed by treatment with 10 μM purmorphamine, or DMSO as a control, for another 24 h. Graph represents the mean value of the firefly luciferase activity relative to Renilla transfection control.


( d ) Endogenous LC3 - II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h , either in the absence or presence of trehalose ( 100 mM ) . Where indicated , bafA1 ( 400 nM ) was added for the last 4 h . Quantification by densitometric analysis relative to actin is shown in the graph .

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

(d) Endogenous LC3-II was detected in cell lysates from cells treated with 10 μM purmorphamine for 24 h, either in the absence or presence of trehalose (100 mM). Where indicated, bafA1 (400 nM) was added for the last 4 h. Quantification by densitometric analysis relative to actin is shown in the graph.


( e ) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions , either in the presence of bafA1 or DMSO as a control , and LC3 - II and actin levels were detected .

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(e) Cells were treated with 10 μM purmorphamine for 24 h under starvation conditions, either in the presence of bafA1 or DMSO as a control, and LC3-II and actin levels were detected.


( f ) HA - HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h . The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph . P - values were calculated by odds ratio .

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(f) HA-HttQ74 construct was transfected into HeLa cells followed by treatment with 10 μM purmorphamine for 24 h. The percentage of transfected cells with aggregates detected by HA immunofluorescence is shown in the graph. P-values were calculated by odds ratio.


( g ) U20S cells stably expressing HaloTag - p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine . After 48 h , cells were lysed , run on a SDS - polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager . Fluorescence was normalized to total protein levels , detected on the same gels by Kryton Fluorescence protein stain . A representative gel and its quantitated normalized levels are shown . In all panels , graphs show mean values and error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - test unless indicated : * * * P0 . 001 ; * * P0 . 01 ; * P0 . 05 . SDS - PAGE , SDS - polyacrylamide gel electrophoresis .

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(g) U20S cells stably expressing HaloTag-p62 were labelled with HaloTag ligand for 15 min and washed out followed by treatment with purmorphamine. After 48 h, cells were lysed, run on a SDS-polyacrylamide gel electrophoresis and fluorescent HaloTag was visualized using a Typhoon 8600 variable mode imager. Fluorescence was normalized to total protein levels, detected on the same gels by Kryton Fluorescence protein stain. A representative gel and its quantitated normalized levels are shown. In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test unless indicated: ***P0.001; **P0.01; *P0.05. SDS-PAGE, SDS-polyacrylamide gel electrophoresis.


( a ) HeLa cells were transiently transfected with pcDNA ( vector ) , Ptch1 or Ptch2 constructs for 48 h . Where indicated , cells were treated with bafA1 for the last 4 h . LC3 - II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph .

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(a) HeLa cells were transiently transfected with pcDNA (vector), Ptch1 or Ptch2 constructs for 48 h. Where indicated, cells were treated with bafA1 for the last 4 h. LC3-II levels were detected by western blotting and levels were quantified by densitometric analysis relative to tubulin and shown in graph.


( b ) HeLa were transfected with Ptch1 or Ptch2 together with HA - HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence . P - values were calculated by odds ratio .

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(b) HeLa were transfected with Ptch1 or Ptch2 together with HA-HttQ74 and the percentage of transfected cells with aggregates was assessed by HA immunofluorescence. P-values were calculated by odds ratio.


( c ) Atg5 + / + or Atg5 − / − MEFs were transfected with GFP - HttQ74 for 48 h , cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence .

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(c) Atg5+/+ or Atg5−/− MEFs were transfected with GFP-HttQ74 for 48 h, cells were fixed and the percentage of transfected cells with aggregates was scored by direct fluorescence.


( d , e ) Endogenous LC3 - II levels were detected in HeLa cells transfected with control , Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h ( d ) Quantification and statistical analysis is shown in graphs .

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with 100 mM trehalose in the last 24 h (d) Quantification and statistical analysis is shown in graphs.


( d , e ) Endogenous LC3 - II levels were detected in HeLa cells transfected with control , Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h ( e ) . Quantification and statistical analysis is shown in

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(d,e) Endogenous LC3-II levels were detected in HeLa cells transfected with control, Ptch1 or Ptch2 siRNA and either left untreated or treated with400 nM bafA1 for 4 h (e). Quantification and statistical analysis is shown in graphs


( f ) siRNA - targeting Ptch2 was transfected into HeLa cells stably expressing mRFP - GFP - LC3 and representative confocal microscopy images are shown . Scale bars represent 26 μm . Percentage of GFP - positive vesicles ( autophagosomes ) , RFP - positive vesicles ( total number of vesicles ) or percentage of autolysosomes ( calculated by subtracting numbers of GFP from RFP vesicles ) , quantified using a Cellomics array scan , was calculated relative to control siRNA - transfected cells . Although an increase in autophagosome biogenesis generally increases both the number of GFP + vesicles and the number of GFP - / RFP + vesicles as a result of an enhanced autophagy flux , defects in autophagosome degradation , such as in bafA1 - treated cells , increase GFP + vesicles but not GFP − / RFP + structures , as GFP remains intact . Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100 . In all panels , unless indicated , graphs show mean values and error bars represent s . d . from a triplicate experiment representative of at least three independent experiments . Statistical analyses were performed by two - tail Student ' s t - test : * * P0 . 01 ; * P0 . 05 ; NS , not significant .

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

(f) siRNA-targeting Ptch2 was transfected into HeLa cells stably expressing mRFP-GFP-LC3 and representative confocal microscopy images are shown. Scale bars represent 26 μm. Percentage of GFP-positive vesicles (autophagosomes), RFP-positive vesicles (total number of vesicles) or percentage of autolysosomes (calculated by subtracting numbers of GFP from RFP vesicles), quantified using a Cellomics array scan, was calculated relative to control siRNA-transfected cells. Although an increase in autophagosome biogenesis generally increases both the number of GFP+ vesicles and the number of GFP-/RFP+ vesicles as a result of an enhanced autophagy flux, defects in autophagosome degradation, such as in bafA1-treated cells, increase GFP+ vesicles but not GFP−/RFP+ structures, as GFP remains intact. Graph shows the mean value obtained from four independent experiments in triplicate and with control conditions set to 100. In all panels, unless indicated, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analyses were performed by two-tail Student's t-test: **P0.01; *P0.05; NS, not significant.


( a ) Smo siRNA - treated cells were transfected with HA - HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA - positive cells by immunofluorescence . Graphs show mean values and error bars represent s . d . of a representative experiment performed in triplicate . P - values were calculated by odds ratio .

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(a) Smo siRNA-treated cells were transfected with HA-HttQ74 for the last 24 h and the percentage of transfected cells with aggregates scored in HA-positive cells by immunofluorescence. Graphs show mean values and error bars represent s.d. of a representative experiment performed in triplicate. P-values were calculated by odds ratio.


( b ) siRNA - targeting Smo was transfected into HeLa cells stably expressing mRFP - GFP - LC3 . Representative confocal microscopy images are shown . Bars represent 26 μm . The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA - transfected cells . Statistical tests were performed on data from three independent experiments in triplicate . * P0 . 05 ; * * * P0 . 001 .

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(b) siRNA-targeting Smo was transfected into HeLa cells stably expressing mRFP-GFP-LC3. Representative confocal microscopy images are shown. Bars represent 26 μm. The number of vesicles was analyzed in a Cellomics array scan and percentage was calculated relative to control siRNA-transfected cells. Statistical tests were performed on data from three independent experiments in triplicate. *P0.05; ***P0.001.


( a ) Wild - type Gli + / + or Gli1 − / − , Gli2 − / − and Gli3 − / − MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total cell lysates were subjected to immunoblot with anti - LC3 and anti - actin antibodies .

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(a) Wild-type Gli+/+ or Gli1−/−, Gli2−/− and Gli3−/− MEFs were treated with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( b ) Gli2 − / − MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a . with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h , or left untreated . Total cell lysates were subjected to immunoblot with anti - LC3 and anti - actin antibodies .

(b) Gli2−/− MEFs were transfected for 48 h with either empty vector or Gli2 DNA construct and treated as in a. with 100 mM trehalose alone or in combination with 10 μM of Pmph for 24 h, or left untreated. Total cell lysates were subjected to immunoblot with anti-LC3 and anti-actin antibodies.


( c ) LC3 - II levels were detected by western blotting of total lysates from wild - type and Gli2 − / − mice embryos ( E12 . 5 ) . For quantification , LC3 - II levels from a total of five wild - type and five Gli2 − / − embryos were normalized to actin , wild - type was set to 100 . Graphs show mean values and error bars represent s . d . Statistical analysis were performed by two - tail Student ' s t - test * * P0 . 01 . Pmph , purmorphamine .

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(c) LC3-II levels were detected by western blotting of total lysates from wild-type and Gli2−/− mice embryos (E12.5). For quantification, LC3-II levels from a total of five wild-type and five Gli2−/− embryos were normalized to actin, wild-type was set to 100. Graphs show mean values and error bars represent s.d. Statistical analysis were performed by two-tail Student's t-test **P0.01. Pmph, purmorphamine.


( a , b ) Control ( a ) or hh [ ts2 ] ( b ) larvae were incubated at permissive temperature ( 18 ° C ) to the third instar stage , then cultured at non - permissive temperature ( 29 ° C ) in well - fed conditions for 24 h . Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed .

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(a,b) Control (a) or hh[ts2] (b) larvae were incubated at permissive temperature (18 °C) to the third instar stage, then cultured at non-permissive temperature (29 °C) in well-fed conditions for 24 h. Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed.


( c ) Lysotracker Red - labelled fat body containing single - cell flipout clones ( GFP positive ) overexpressing patched . Fat body was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI and imaged unfixed .

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(c) Lysotracker Red-labelled fat body containing single-cell flipout clones (GFP positive) overexpressing patched. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( d ) Confocal image of a GFP - positive patched - expressing clone , with mCherry - Atg8a expressed in all cells . Fat body was dissected from well - fed larvae and imaged after formaldehyde fixation .

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(d) Confocal image of a GFP-positive patched-expressing clone, with mCherry-Atg8a expressed in all cells. Fat body was dissected from well-fed larvae and imaged after formaldehyde fixation.


( e ) GFP - marked flipout clones of CiCell - expressing cells . Fat body was dissected from well - fed larvae , incubated in Lysotracker Red / DAPI and imaged unfixed .

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(e) GFP-marked flipout clones of CiCell-expressing cells. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed.


( f ) Confocal image of fixed mCherry - Atg8a expressing fat body containing a GFP - marked clone of CiCell - expressing cells .

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(f) Confocal image of fixed mCherry-Atg8a expressing fat body containing a GFP-marked clone of CiCell-expressing cells.


( g ) Cells homozygous for the null costal2 [ 2 ] allele ( marked by lack of GFP ) were generated by flp / FRT - mediated recombination in mCherry - Atg8a expressing fat body . Larvae were starved 4 h before dissection and fixation .

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(g) Cells homozygous for the null costal2[2] allele (marked by lack of GFP) were generated by flp/FRT-mediated recombination in mCherry-Atg8a expressing fat body. Larvae were starved 4 h before dissection and fixation.


( a ) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells . Each circle represents an autophagy gene ; those with largest fold - changes are indicated in red . The central line represents no changes in expression ; above the central line , genes whose expression is increased : below , those with reduced levels . Grey lines indicate 2 - fold increase or decrease . ( b ) Table showing the 10 genes with higher fold changes in expression and the values obtained .

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(a) Graph representing fold change in mRNA levels in cells treated with Shh against untreated cells. Each circle represents an autophagy gene; those with largest fold-changes are indicated in red. The central line represents no changes in expression; above the central line, genes whose expression is increased: below, those with reduced levels. Grey lines indicate 2-fold increase or decrease. (b) Table showing the 10 genes with higher fold changes in expression and the values obtained.


( c ) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h . Actin was used as a protein loading control .

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(c) Levels of PERK protein were assessed by western blotting in HeLa cells treated with 10 μM purmorphamine for 24 h. Actin was used as a protein loading control.


( d ) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h . Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI - COR infra - red imager .

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(d) HeLa cells were treated with purmorphamine for 24 h and either left in rich media or in HBSS for the last 4 h. Levels of S51 phoshorylation and total levels of eIF2α were detected by western blotting using LI-COR infra-red imager.


( e , f ) Wild - type and PERK − / − ( e ) or eIF2α S51A / S51A MEFs ( f ) were treated with DMSO or 10 μM purmorphamine for 24 h and , where indicated , cells were treated with bafA1 for the last 4 h . tubulin - II and tubulin or tubulin , as a loading control , were detected and a representative blot is shown . HBSS , Hank ' s balanced salt solution .

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(e,f) Wild-type and PERK−/− (e) or eIF2α S51A/S51A MEFs (f) were treated with DMSO or 10 μM purmorphamine for 24 h and, where indicated, cells were treated with bafA1 for the last 4 h. tubulin-II and tubulin or tubulin, as a loading control, were detected and a representative blot is shown. HBSS, Hank's balanced salt solution.


( A ) Representative images of a PDGFB tumour : H & E on the right to identify the tumour , an adjacent section stained for Iba1 ( red ) , p - S6 ( green ) and DAPI ( blue ) at the centre and the region selection for quantification on the left ( non - tumour in light blue , tumour edge in orange and tumour core in dark blue ) .

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(A) Representative images of a PDGFB tumour: H&E on the right to identify the tumour, an adjacent section stained for Iba1 (red), p-S6 (green) and DAPI (blue) at the centre and the region selection for quantification on the left (non-tumour in light blue, tumour edge in orange and tumour core in dark blue).


( B ) Representative images of core , edge and adjacent non - tumour brain tissue for Iba1 , p - S6 and DAPI staining in Pten - / - p53 - / - ( right ) and GL261 ( left ) tumours . Percentage of Iba1 + cells co - expressing p - S6 in the three defined regions . Five high grade glioma models were analysed - GL261 ( n = 3 ) , PDGFB ( n = 3 ) , Ntv - a ; PDGFB + shp53 ( n = 2 ) , Pten - / - p53 - / - ( n = 3 ) , and Pten - / - p53 - / - Idh1mut ( n = 3 ) ( mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(B) Representative images of core, edge and adjacent non-tumour brain tissue for Iba1, p-S6 and DAPI staining in Pten-/-p53-/- (right) and GL261 (left) tumours. Percentage of Iba1+ cells co-expressing p-S6 in the three defined regions. Five high grade glioma models were analysed - GL261 (n=3), PDGFB (n=3), Ntv-a;PDGFB+shp53 (n=2), Pten-/-p53-/- (n=3), and Pten-/-p53-/-Idh1mut (n=3) (mean ±SEM; Two-Way ANOVA Tukey test).


( F , G ) Expression levels of ( F ) Rps6 and ( G ) Eif4ebp1 in healthy versus GL261 microglia and macrophage ( n = 3 ; mean ± SEM , likelihood ratio test in edgeR ) .

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(F,G) Expression levels of (F) Rps6 and (G) Eif4ebp1 in healthy versus GL261 microglia and macrophage (n=3; mean ±SEM, likelihood ratio test in edgeR).


Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr ( B and normalised against non - phosphorylated protein and vinculin analysed on the same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr (B and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( C ) p - S6 S240 / 244 Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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Flow cytometry analysis was carried out in microglia for (C) p-S6 S240/244 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr and normalised against non - phosphorylated protein and vinculin analysed on the same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( E ) p - 4EBP1 T37 / 46 Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

Flow cytometry analysis was carried out in microglia for (E) p-4EBP1 T37/46 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( G ) p - AKT S473 ; Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin , was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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Flow cytometry analysis was carried out in microglia for (G) p-AKT S473; Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin, was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 0 . 5hr ( H ) and normalised against non - phosphorylated protein and vinculin analysed on the same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + LY ) was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 0.5hr (H) and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM +LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( I ) p - AKT T308 . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + LY ) was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

Flow cytometry analysis was carried out in microglia for (I) p-AKT T308. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+ LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


( B ) Survival analysis for Cx3cr1 - Rheb1Δ / Δ ( n = 7 ) and Rheb1fl / fl ( n = 7 ) mice . Chi square test .

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(B) Survival analysis for Cx3cr1-Rheb1Δ/Δ (n=7) and Rheb1fl/fl (n=7) mice. Chi square test.


( C ) Representative images and quantification of the tumour volume with MRI of Rheb1fl / fl ( n = 8 ) compared to Cx3cr1 - Rheb1Δ / Δ ( n = 8 ) mice . ( mean ± SEM ; Unpaired parametric t test ) .

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(C) Representative images and quantification of the tumour volume with MRI of Rheb1fl/fl (n=8) compared to Cx3cr1-Rheb1Δ/Δ (n=8) mice. (mean ±SEM; Unpaired parametric t test).


( D ) H & E staining showing representative histological features ( overview and high magnification of microvascular proliferation ) of Rheb1fl / fl and Cx3cr1 - Rheb1Δ / Δ GL261 tumours . Scale bar is 125 μm ( top , H & E ) and 80 μm ( all other images )

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(D) H&E staining showing representative histological features (overview and high magnification of microvascular proliferation) of Rheb1fl/fl and Cx3cr1-Rheb1Δ/Δ GL261 tumours. Scale bar is 125 μm (top, H&E) and 80 μm (all other images)


( E ) Percentage of GFP + CD45 - GL261 tumour cells in Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) and Rheb1fl / fl ( n = 7 ) tumours , with representative FACS plot ( mean ± SEM ; Unpaired parametric t test ) .

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(E) Percentage of GFP+ CD45- GL261 tumour cells in Cx3cr1-Rheb1Δ/Δ (n=5) and Rheb1fl/fl (n=7) tumours, with representative FACS plot (mean ±SEM; Unpaired parametric t test).


( B ) Levels of TAM - MG ( P2RY12 + CD49d - ) and TAM - BMDM ( P2RY12 - CD49d + ) gated from CD45 + population in GL261 tumours from Rheb1fl / fl ( n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) mice by flow cytometry ( representative flow cytometry plots on top and quantification at the bottom ; mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(B) Levels of TAM-MG (P2RY12+ CD49d-) and TAM-BMDM (P2RY12- CD49d+) gated from CD45+ population in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice by flow cytometry (representative flow cytometry plots on top and quantification at the bottom; mean ±SEM; Two-Way ANOVA Tukey test).


( C ) Percentage of CD8 CTL ( top ) and CD4 Th ( CD4 ) cells in GL261 tumours from Rheb1fl / fl ( n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) mice assessed by flow cytometry ( representative flow cytometry plots , left and quantification , right ; mean ± SEM ; Unpaired t test ) .

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(C) Percentage of CD8 CTL (top) and CD4 Th (CD4) cells in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice assessed by flow cytometry (representative flow cytometry plots, left and quantification, right; mean ±SEM; Unpaired t test).


( D ) Tumour tissues from Rheb1fl / fl ( n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( n = 4 ) were stained for CD8 , CD4 and FoxP3 . DAB staining was quantified as % of positive cells using Definiens software . Representative images of the stained tissues are shown . ( mean ± SEM ; unpaired parametric t test ) . Scale bar is 100µm .

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(D) Tumour tissues from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=4) were stained for CD8, CD4 and FoxP3. DAB staining was quantified as % of positive cells using Definiens software. Representative images of the stained tissues are shown. (mean ±SEM; unpaired parametric t test). Scale bar is 100µm.


( B ) Heatmap of the differential correlation analysis between CIBERSORT cell fractions and signatures enrichment scores ( ssGSEA ) for lymphocytes in the Cx3cr1 - Rheb1Δ / Δ ( n = 3 ) vs Rheb1fl / fl ( n = 3 ) GL261 tumours .

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(B) Heatmap of the differential correlation analysis between CIBERSORT cell fractions and signatures enrichment scores (ssGSEA) for lymphocytes in the Cx3cr1-Rheb1Δ/Δ (n=3) vs Rheb1fl/fl (n=3) GL261 tumours.


( C , D ) CD4 Th cells ( C ) and CD8 CTL cells ( D ) expression of Ki67 , perforin , granzyme b , and IFNγ , in Cx3cr1 - Rheb1fl / fl ( n = 5 ) and Rheb1Δ / Δ ( n = 5 ) GL261 tumours . Representative FACS plots show control samples ( tumour - derived cells stimulated in culture in the absence of protein inhibitor cocktail - in orange ) compared to stimulated cells derived from Rheb1fl / fl ( fl / fl , red ) and Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , blue ) GL261 tumours ( cells stimulated in culture with protein inhibitor cocktail ) ( mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(C,D) CD4 Th cells (C) and CD8 CTL cells (D) expression of Ki67, perforin, granzyme b, and IFNγ, in Cx3cr1-Rheb1fl/fl (n=5) and Rheb1Δ/Δ (n=5) GL261 tumours. Representative FACS plots show control samples (tumour-derived cells stimulated in culture in the absence of protein inhibitor cocktail - in orange) compared to stimulated cells derived from Rheb1fl/fl (fl/fl, red) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, blue) GL261 tumours (cells stimulated in culture with protein inhibitor cocktail) (mean ±SEM; Two-Way ANOVA Tukey test).


( E , F ) Expression of CD44 ( E ) and CD62L ( F ) by CD4 Th and CD8 CTL in Cx3cr1 - Rheb1fl / fl ( n = 5 ; fl / fl , red ) and Rheb1Δ / Δ ( n = 5 ; Δ / Δ , blue ) GL261 tumours with representative FACS plots show control samples ( mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(E,F) Expression of CD44 (E) and CD62L (F) by CD4 Th and CD8 CTL in Cx3cr1-Rheb1fl/fl (n=5; fl/fl, red) and Rheb1Δ/Δ (n=5; Δ/Δ, blue) GL261 tumours with representative FACS plots show control samples (mean ±SEM; Two-Way ANOVA Tukey test).


( B ) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change ( Log ( FC ) ) .

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(B) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change (Log(FC)).


( C ) Upstream regulators as identified by the IPA software from differentially regulated genes .

(C) Upstream regulators as identified by the IPA software from differentially regulated genes.


( E ) Microglia were pre - treated with Torin , or medium as indicated and then stimulated with mNSC - CM , mGICPten - / - ; p53 - / - - CM , or GL261 - CM . p - NF - ĸB ( p - P65 ) and p - STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr .

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(E) Microglia were pre-treated with Torin, or medium as indicated and then stimulated with mNSC-CM, mGICPten-/-;p53-/--CM, or GL261-CM. p-NF-ĸB (p-P65) and p-STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr.


( F ) Immunofluorescence images of NF - ĸB ( P65 ) in conditioned microglia . Quantification of nuclear translocation of NF - κB from staining . Units represent the % of voxels in the NF - κB channel colocalised with DAPI ( n = 3 ; mean ± SEM ; One - Way ANOVA Tukey test - right ) . Scale bar is 50µm .

(F) Immunofluorescence images of NF-ĸB (P65) in conditioned microglia. Quantification of nuclear translocation of NF-κB from staining. Units represent the % of voxels in the NF-κB channel colocalised with DAPI (n=3; mean ±SEM; One-Way ANOVA Tukey test - right). Scale bar is 50µm.


( G ) Production of Il - 12 ( Il - 12p40 ) , Tnf , Il6 , and Il10 by conditioned microglia was determined by qPCR . Each treatment was normalised to housekeeping gene and the unconditioned control . ( n = 3 ; mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(G) Production of Il-12 (Il-12p40), Tnf, Il6, and Il10 by conditioned microglia was determined by qPCR. Each treatment was normalised to housekeeping gene and the unconditioned control. (n=3; mean ±SEM; Two-Way ANOVA Tukey test).


( H , I ) MFI levels of pSTAT3 in TAM - MG ( CD45 + P2RY12 + CD49d - ) ( H ) and p - NF - κB ( p - P65 ) in TAM - BMDM ( CD45 + P2RY12 - CD49d + ) ( I ) from Rheb1fl / fl ( fl / fl , blue , n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , red , n = 5 ) GL261 tumours . Representative FACS plots are displayed for each cell type ( mean ± SEM , Two - Way ANOVA Tukey test ) .

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(H,I) MFI levels of pSTAT3 in TAM-MG (CD45+ P2RY12+ CD49d-) (H) and p-NF-κB (p-P65) in TAM-BMDM (CD45+ P2RY12- CD49d+) (I) from Rheb1fl/fl (fl/fl, blue, n=4) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, red, n=5) GL261 tumours. Representative FACS plots are displayed for each cell type (mean ±SEM, Two-Way ANOVA Tukey test).


( A ) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM - MG or TAM - BMDM signatures in TCGA GBM transcriptomic data . Comparison carried out on all IDH - wild - type samples , and in a subgroup specific manner according to Wang ' s classifier . Size of circle is indicative of Rsquare value and bold outline represents a p - value ≤ 0 . 05 .

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(A) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM-MG or TAM-BMDM signatures in TCGA GBM transcriptomic data. Comparison carried out on all IDH-wild-type samples, and in a subgroup specific manner according to Wang's classifier. Size of circle is indicative of Rsquare value and bold outline represents a p-value ≤0.05.


( B ) Separation of IDH - wildtype GBM samples between those displaying high mTOR and microglia enrichment ( + ve correlation , group 1 in orange ) and those without this signature ( group 2 in green ) .

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(B) Separation of IDH-wildtype GBM samples between those displaying high mTOR and microglia enrichment (+ve correlation, group 1 in orange) and those without this signature (group 2 in green).


( C ) CIBERSORT cell fractions calculated from the TPM of TCGA IDH - wild type GBM samples from group 1 compared to those from group 2 .

(C) CIBERSORT cell fractions calculated from the TPM of TCGA IDH-wild type GBM samples from group 1 compared to those from group 2.


( F ) Staining by flow cytometry of EPSC , YS - EBs and matched - iMGL with indicated markers ( n = 3 ; mean ± SEM ) .

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(F) Staining by flow cytometry of EPSC, YS-EBs and matched-iMGL with indicated markers (n=3; mean ±SEM).


( G , H ) Mature iMGL were immunostained for ( G ) PU1 and CD11b and ( H ) Iba1 and TREM2 . Scale bar 20µm .

(G,H) Mature iMGL were immunostained for (G) PU1 and CD11b and (H) Iba1 and TREM2. Scale bar 20µm.