F , G . Western blotting for TRF2 expression in Telomerase Positive ( HeLa , HCT116 , MDA - MB - 231 , MDA - MB - 436 ) and ALT positive ( U2 - OS , Saos - 2 ) cells transiently transfected with miR - Control or miR - 182 - 3p . Upper panel shows the quantification of TRF2 expression . Bottom panel , representative images are shown , actin was used as loading control . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney test .

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

F,G. Western blotting for TRF2 expression in Telomerase Positive (HeLa, HCT116, MDA-MB-231, MDA-MB-436) and ALT positive (U2-OS, Saos-2) cells transiently transfected with miR-Control or miR-182-3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


H . U2 - OS cells transiently transfected with the miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post - transfection . Left panel , representative images . Scale bar : 10µm . Right panel , quantification of TRF2 fluorescence intensity . a . f . u . arbitrary fluorescence units . N = number of analysed nuclei . Red bar indicates mean value . Data information : Three independent experiments were performed ( n = 3 ) . P values are determined by Student ' s t - test ; for ( H )

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

H. U2-OS cells transiently transfected with the miR-Control, miR-182-3p or miR-182-3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post-transfection. Left panel, representative images. Scale bar: 10µm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N=number of analysed nuclei. Red bar indicates mean value. Data information: Three independent experiments were performed (n=3). P values are determined by Student's t-test; for (H)


I . U2 - OS cells transfected as described in ( H ) were assayed by immuno - fluorescence combined with telomeric FISH . Left panel , representative images of co - localizations between TRF2 and telomeres ( white arrowheads ) . Scale bar : 10µm . Right panel , co - localizations were analysed using imageJ software . N = number of analysed nuclei . Data information : For I , data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney test .

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

I. U2-OS cells transfected as described in (H) were assayed by immuno-fluorescence combined with telomeric FISH. Left panel, representative images of co-localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10µm. Right panel, co-localizations were analysed using imageJ software. N=number of analysed nuclei. Data information: For I, data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


A . Immunofluorescence analysis of γH2AX combined with telomeric FISH ( TIFs ) was performed in MDA - MB - 231 cells transfected with the indicated miRNAs or siRNAs . The mean number of TIFs per nucleus was analysed . B . Representative images and enlargements of co - localizations ( white arrowheads ) relative to the experiment described in A . Scale bar : 10µm . Data information : For A data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated miRNAs or siRNAs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analysed. B. Representative images and enlargements of co-localizations (white arrowheads) relative to the experiment described in A. Scale bar: 10µm. Data information: For A data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs or siRNAs.


C . Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe ( PIFs ) was performed in MDA - MB - 231 cells transfected with the indicated miRNAs or siRNAs . The γH2AX positive cells with ≥ 1 PIFs per nucleus were analysed . D . Representative images of co - localizations ( white arrowheads ) relative to the experiment described in C . Scale bar : 10µm . Data information : For C , data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated miRNAs or siRNAs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed. D. Representative images of co-localizations (white arrowheads) relative to the experiment described in C. Scale bar: 10µm. Data information: For C, data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs or siRNAs.


E . Quantification of TIFs in MDA - MB - 231 cells over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . The mean number of TIFs per nucleus was quantified . F . Representative images and enlargements relative to the experiment described in E . White arrowheads indicate co - localizations events . Scale bar : 10µm . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated

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

E. Quantification of TIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. F. Representative images and enlargements relative to the experiment described in E. White arrowheads indicate co-localizations events. Scale bar: 10µm. Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs


G . Quantification of PIFs in MDA - MB - 231 cells over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . The γH2AX positive cells with ≥ 1 PIFs per nucleus were analysed . H . Representative images relative to the experiment described in G . White arrowheads indicate co - localizations events . Scale bar : 10µm . Data information : For G data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated

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

G. Quantification of PIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed. H. Representative images relative to the experiment described in G. White arrowheads indicate co-localizations events. Scale bar: 10µm. Data information: For G data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs


A , B . MDA - MB - 436 and MDA - MB - 231 cells underwent two rounds of transfection with miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor . Starting from the day of the second transfection , cell confluence was monitored by Incucyte every 24h up to a maximum of 3 days . The percentage of cell confluence was analysed . Data information : For A , B data are shown as mean ± SEM . three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test .

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

A,B. MDA-MB-436 and MDA-MB-231 cells underwent two rounds of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24h up to a maximum of 3 days. The percentage of cell confluence was analysed. Data information: For A,B data are shown as mean ± SEM. three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test.


C , D . Cell number of MDA - MB - 436 ( C ) and MDA - MB - 231 ( D ) cells and TRF2 expression were analysed by automatic cell count and by western blotting at the end of the experiment described in A and B . Actin was used as loading control . Data information : For C , D data are shown as mean ± SD . three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test .

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

C,D. Cell number of MDA-MB-436 (C) and MDA-MB-231 (D) cells and TRF2 expression were analysed by automatic cell count and by western blotting at the end of the experiment described in A and B. Actin was used as loading control. Data information: For C,D data are shown as mean ± SD. three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test.


E . Two - dimensional scatter plots of Annexin V analysis performed in MDA - MB - 436 at the end of the second cycle of transfection with miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor . Red boxes indicate early and late apoptotic cells . F . Quantification of Annexin V positive cells ( % ) of experiment described in E . G . Two - dimensional scatter plots of Annexin V analysis performed in MDA - MB - 231 as described in E . H . Quantification of Annexin V positive cells ( % ) of experiment described in G . Data information : For F , H data are shown as mean ± SD . P values are determined by unpaired two - tailed t test . For F , H two different biological replicates were performed .

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

E. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-436 at the end of the second cycle of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Red boxes indicate early and late apoptotic cells. F. Quantification of Annexin V positive cells (%) of experiment described in E. G. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-231 as described in E. H. Quantification of Annexin V positive cells (%) of experiment described in G. Data information: For F,H data are shown as mean ± SD. P values are determined by unpaired two-tailed t test. For F, H two different biological replicates were performed.


I , J . MDA - MB - 436 cells over - expressing TRF2 or an empty vector ( pBabe ) were transiently transfected with indicated miRNAs and cell count ( I ) or apoptosis ( J ) analysis was performed 72h post - transfection . Data information : For , I , J , data are shown as mean ± SD . For I , three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . For J , two different biological replicates were performed .

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

I,J. MDA-MB-436 cells over-expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72h post-transfection. Data information: For ,I,J, data are shown as mean ± SD. For I, three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. For J, two different biological replicates were performed.


A . Western blotting for TRF2 expression in BJ cells transiently transfected with miR - Control or miR - 182 - 3p . The graph represents the quantification of three independent experiments . Representative images are shown , actin was used as loading control . Unspecific bands are indicated with ( * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blotting for TRF2 expression in BJ cells transiently transfected with miR-Control or miR-182-3p. The graph represents the quantification of three independent experiments. Representative images are shown, actin was used as loading control. Unspecific bands are indicated with (*).


B , C . Mean of γH2AX foci per nucleus was analysed in BJ cells 72h post - transfection with the indicated miRNAs . Representative images of γH2AX foci are shown in C . Data information : student t test was used to calculate statistical significance . Scale bars ( 10µm ) . P values are indicated .

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

B, C. Mean of γH2AX foci per nucleus was analysed in BJ cells 72h post-transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in C. Data information: student t test was used to calculate statistical significance. Scale bars (10µm). P values are indicated.


D . Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe ( TIFs ) was performed in BJ cells 72h post - transfection with the indicated miRNAs . Left panel : the mean number of TIFs per nucleus was analysed . Right panel : representative images and enlargements of co - localizations . Data information : a student t test was used to calculate statistical significance . Scale bars ( 10µm ) . P values are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72h post-transfection with the indicated miRNAs. Left panel: the mean number of TIFs per nucleus was analysed. Right panel: representative images and enlargements of co-localizations. Data information: a student t test was used to calculate statistical significance. Scale bars (10µm). P values are indicated.


E . Cells number of BJ cells was analysed by automatic cell count at the end of the second round of transfection with miR - Control or miR - 182 - 3p . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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

E. Cells number of BJ cells was analysed by automatic cell count at the end of the second round of transfection with miR-Control or miR-182-3p. Data information: a student t test was used to calculate statistical significance. P values are indicated.


F . FACS analysis to evaluate cell cycle progression by Propidium Iodide ( PI ) staining in BJ cells treated as indicated in E .

O 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. FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in E.


G . β - Galactosidase assay in BJ cells after two rounds of transfection with mimic miR - Control or miR - 182 - 3p . Left panel : analysis of β - Galactosidase positive cells . Right panel : representative images . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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

G. β-Galactosidase assay in BJ cells after two rounds of transfection with mimic miR-Control or miR-182-3p. Left panel: analysis of β-Galactosidase positive cells. Right panel: representative images. Data information: a student t test was used to calculate statistical significance. P values are indicated.


H - J . IL - 6 ( H ) , CXCL1 ( I ) , IL - 8 ( J ) factors were analysed by ELISA to evaluated the Senescence - associated secretory phenotype ( SASP ) in BJ cells treated as indicated in G . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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

H-J. IL-6 (H), CXCL1 (I), IL-8 (J) factors were analysed by ELISA to evaluated the Senescence-associated secretory phenotype (SASP) in BJ cells treated as indicated in G. Data information: a student t test was used to calculate statistical significance. P values are indicated.


A , B . MDA - MB - 231 ( A ) and MDA - MB - 436 ( B ) tumor xenografts were treated with LNPs - empty , LNPs - miR - Control or by LNPs - miR - 182 - 3p when the tumors became palpable . Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs - miR - Control , LNPs - miR - 182 - 3p or equivalent volume of LNPs - empty as indicated in the scheduling . The mean of tumor volumes ( n = 5 per group ) is shown . Data information : For A , B data are shown as mean ± SD . P values are determined by unpaired two - tailed t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MDA-MB-231 (A) and MDA-MB-436 (B) tumor xenografts were treated with LNPs-empty, LNPs-miR-Control or by LNPs-miR-182-3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs-miR-Control, LNPs-miR-182-3p or equivalent volume of LNPs-empty as indicated in the scheduling. The mean of tumor volumes (n=5 per group) is shown. Data information: For A, B data are shown as mean ± SD. P values are determined by unpaired two-tailed t test


C , D . Tumors from mice treated in A and B were processed to measure miR - 182 - 3p expression by TaqMan qPCR . Data information : For C , D the line in the middle of the box plot denote a median value , the limits of box represent the interquartile range ( 25th to 75th percentiles ) , while , the whiskers denote the minimum to maximum values . , P values are determined by unpaired two - tailed t test ;

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

C,D. Tumors from mice treated in A and B were processed to measure miR-182-3p expression by TaqMan qPCR. Data information: For C, D the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. , P values are determined by unpaired two-tailed t test;


E . Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA - MB - 231 human breast cancer xenografts . Scale bar : 50μm . F . The histograms show : the expression of TRF2 , calculated as immunoreactivity score ( IRS ) by IHC , and the count of positive cells to γH2AX , Tunel or CD31 staining . The analyses were performed on 3 mice per group , the points represent the number of field analysed for each condition . Data information : For F , data are shown as mean ± SD . ( F ) , P values are determined by Mann - Whitney test .

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

E. Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA-MB-231 human breast cancer xenografts. Scale bar: 50μm. F. The histograms show: the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, Tunel or CD31 staining. The analyses were performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For F, data are shown as mean ± SD. (F), P values are determined by Mann-Whitney test.


G , H . Luminescent MDA - MB - 436 cells were injected into the brain and monitored by IVIS imaging system . After one week from implant , treatment with LNPs - miR - Control and LNPs - miR - 182 - 3p was performed as indicated in A and B . Representative images from in vivo ( upper panel ) or ex - vivo ( bottom panel ) brain tumors are shown in G . Boxplots ( H ) show the measurement of photons for each brain tumor ( n = 5 per group ) acquired at the indicated times . Data information : For H , the line in the middle of the box plot denote a median value , the limits of box represent the interquartile range ( 25th to 75th percentiles ) , while , the whiskers denote the minimum to maximum values . For H , P values are determined by unpaired two - tailed t test ;

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

G, H. Luminescent MDA-MB-436 cells were injected into the brain and monitored by IVIS imaging system. After one week from implant, treatment with LNPs-miR-Control and LNPs-miR-182-3p was performed as indicated in A and B. Representative images from in vivo (upper panel) or ex-vivo (bottom panel) brain tumors are shown in G. Boxplots (H) show the measurement of photons for each brain tumor (n=5 per group) acquired at the indicated times. Data information: For H, the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. For H, P values are determined by unpaired two-tailed t test;


A , B . PDTCs # 1 and # 2 underwent two rounds of transfection with miR - Control or miR - 182 - 3p . Three days after the second transfection , miR - 182 - 3p and TRF2 expression were analysed by TaqMan qPCR and western blotting , respectively . Actin was used as loading control . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t test For the experiments showed in A , B two or three biological replicates were performed , respectively .

A,B. PDTCs #1 and #2 underwent two rounds of transfection with miR-Control or miR-182-3p. Three days after the second transfection, miR-182-3p and TRF2 expression were analysed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in A, B two or three biological replicates were performed, respectively.


C , D . Left panel , area of each PDTCs was measured by ImageJ . Right panel , representative images are shown . Scale bar : 50μm . At least 85 3D cells were analysed for each experimental condition . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t test For the experiments showed in C , D two or three biological replicates were performed , 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 O O O O O O O O O O O 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,D. Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50μm. At least 85 3D cells were analysed for each experimental condition. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in C, D two or three biological replicates were performed, respectively.


E . NSG mice implanted with breast PDTX ( # 2 ) were treated with LNPs - empty , LNPs - miR - Control or LNPs - miR - 182 - 3p as indicated in the scheduling . Caliper measurement of tumors were taken at the indicated days . The mean of tumor volumes ( n = 5 per group ) is shown . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t test ;

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

E. NSG mice implanted with breast PDTX (#2) were treated with LNPs-empty, LNPs-miR-Control or LNPs-miR-182-3p as indicated in the scheduling. Caliper measurement of tumors were taken at the indicated days. The mean of tumor volumes (n=5 per group) is shown. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test;


F . miR - 182 - 3p expression of tumors from mice treated in ( E ) was assayed by TaqMan qPCR . Data information : data are shown as mean ± SD . P values are determined by unpaired two - tailed t

O O O O O O O O O O O O O O O O O O O O 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. miR-182-3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test


G . Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in E . Scale bar : 50μm . H . The histograms show : the expression levels of TRF2 measured as immunoreactivity score ( IRS ) , the percentage of positive cells to γH2AX and Tunel . The analysis was performed on 3 mice per group , the points represent the number of field analysed for each condition . Data information : For H , data are shown as mean ± SD . For ( H ) , P values are determined by Mann - Whitney test .

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

G. Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in E. Scale bar: 50μm. H. The histograms show: the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and Tunel. The analysis was performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For H, data are shown as mean ± SD. For (H), P values are determined by Mann-Whitney test.


A . Representative images of intestine sections from mice previously treated with LNPs - Empty or LNPs - miR - 182 - 3p . H & E staining ( scale bar : 200μm ) and IHC analysis with TRF2 or γH2AX antibodies are shown ( scale bar : 50μm ) . B , C . Quantification of TRF2 expression as immunoreactivity score ( IRS ) ( B ) and of γH2AX positive cells ( % ) ( C ) on intestine samples . D . Representative H & E ( scale bar : 200μm ) , TRF2 and γH2AX images of skin samples corresponding to LNPs - Empty or LNPs - miR - 182 - 3p treated animals ( scale bar : 50μm ) . E , F . Quantification of TRF2 expression as immunoreactivity score ( IRS ) ( E ) and of γH2AX positive cells ( % ) ( F ) on skin samples . G . Representative H & E ( scale bar : 200μm ) , TRF2 and γH2AX images of bone marrow samples corresponding to LNPs - Empty or LNPs - miR - 182 - 3p treated animals ( scale bar : 50μm ) . H , I . Quantification of TRF2 expression as immunoreactivity score ( IRS ) ( H ) and of γH2AX positive cells ( % ) ( I ) on bone marrow samples . Data information : For B , C , E , F , H , I , data are shown as mean ± SD . A Mann - Whitney test t test was used to calculate statistical significance . Four mice per group were analysed the points represent the number of field analysed for each condition .

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

A. Representative images of intestine sections from mice previously treated with LNPs-Empty or LNPs-miR-182-3p. H&E staining (scale bar: 200μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50μm). B,C. Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX positive cells (%) (C) on intestine samples. D. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of skin samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). E,F. Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX positive cells (%) (F) on skin samples. G. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). H,I. Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX positive cells (%) (I) on bone marrow samples. Data information: For B,C,E,F,H,I, data are shown as mean ± SD. A Mann-Whitney test t test was used to calculate statistical significance. Four mice per group were analysed the points represent the number of field analysed for each condition.


( B ) A fragment encompassing amino acids 432 - 520 of Atg13 ( wt ) or the corresponding FV mutant ( FV ) was expressed in E . coli and incubated in excess with an immobilized GST ‐ tagged fragment of Atg1 ( amino acids 501 - 897 ) purified from E . coli ( GST ‐ Atg1 ) . Input ( 5 . 5 % ) and bound proteins were analysed by coomassie blue staining , which stains proteins proportional to their size . The size of marker proteins ( in kDa ) is indicated on the left .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A fragment encompassing amino acids 432-520 of Atg13 (wt) or the corresponding FV mutant (FV) was expressed in E. coli and incubated in excess with an immobilized GST‐tagged fragment of Atg1 (amino acids 501-897) purified from E. coli (GST‐Atg1). Input (5.5%) and bound proteins were analysed by coomassie blue staining, which stains proteins proportional to their size. The size of marker proteins (in kDa) is indicated on the left.


( C ) ATG1 ‐ TAP atg13Δ cells containing endogenously GFP ‐ tagged Atg17 , Atg29 or Vac8 and wild ‐ type ( wt ) or the Atg13 FV ‐ mutant ( F468A ; V469A ) were grown to mid log phase . Atg1 was immunoprecipitated and its association with Atg13 and the GFP ‐ tagged proteins was analysed by immunoblotting . Extract inputs are shown in Supplementary Figure S1B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ATG1‐TAP atg13Δ cells containing endogenously GFP‐tagged Atg17, Atg29 or Vac8 and wild‐type (wt) or the Atg13 FV‐mutant (F468A; V469A) were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 and the GFP‐tagged proteins was analysed by immunoblotting. Extract inputs are shown in Supplementary Figure S1B.


( D ) atg1Δ and atg1Δatg13Δ cells expressing HA ‐ tagged Atg1 and wild ‐ type ( wt ) cells containing an empty plasmid were grown to mid log phase ( rich ) , and then treated with rapamycin ( rapa ) or starved for 4 h in SD ‐ N medium ( starv ) . Atg1 was immunoprecipitated and its association with Atg13 analysed by immunoblotting . Note that phosphorylated Atg13 isolated from cells grown in rich medium migrates slightly slower on SDS - PAGE , and the extent corresponds to the shift typically observed under our gel conditions .

O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) atg1Δ and atg1Δatg13Δ cells expressing HA‐tagged Atg1 and wild‐type (wt) cells containing an empty plasmid were grown to mid log phase (rich), and then treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1 was immunoprecipitated and its association with Atg13 analysed by immunoblotting. Note that phosphorylated Atg13 isolated from cells grown in rich medium migrates slightly slower on SDS-PAGE, and the extent corresponds to the shift typically observed under our gel conditions.


( E ) Yeast cells containing endogenously tagged Atg1 ‐ TAP with and without endogenously tagged Atg13 ‐ GFP , or conversely cells containing endogenously tagged Atg13 ‐ TAP and Atg1 ‐ GFP expressed from a centromeric plasmid were grown to mid log phase ( rich ) and treated with rapamycin ( rapa ) or starved for 4 h in SD ‐ N medium ( starv ) . Atg1 ‐ TAP and Atg13 ‐ TAP were affinity purified and their association with GFP ‐ tagged proteins was analysed by western blotting .

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

(E) Yeast cells containing endogenously tagged Atg1‐TAP with and without endogenously tagged Atg13‐GFP, or conversely cells containing endogenously tagged Atg13‐TAP and Atg1‐GFP expressed from a centromeric plasmid were grown to mid log phase (rich) and treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1‐TAP and Atg13‐TAP were affinity purified and their association with GFP‐tagged proteins was analysed by western blotting.


( F ) Histone3 ‐ HA tagged Atg1 was expressed together with wild ‐ type ( wt ) or the Atg13 ‐ FV mutant fused to Suv methylase ( Suv ) in atg1Δatg13Δ cells , or for control together with Pbs2 ‐ Suv in atg1Δ cells . Logarithmically growing cultures were treated with rapamycin ( rapa ) for 0 or 120 min , and methylation was monitored by preparing cell extracts and western blotting with an anti ‐ trimethylation ‐ specific antibody . Note that Suv ‐ , GFP ‐ or TAP ‐ tagged Atg13 are fully functional , however , do not show the characteristic phosphorylation ‐ induced reduced mobility observed with the untagged protein under our gel conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histone3‐HA tagged Atg1 was expressed together with wild‐type (wt) or the Atg13‐FV mutant fused to Suv methylase (Suv) in atg1Δatg13Δ cells, or for control together with Pbs2‐Suv in atg1Δ cells. Logarithmically growing cultures were treated with rapamycin (rapa) for 0 or 120 min, and methylation was monitored by preparing cell extracts and western blotting with an anti‐trimethylation‐specific antibody. Note that Suv‐, GFP‐ or TAP‐tagged Atg13 are fully functional, however, do not show the characteristic phosphorylation‐induced reduced mobility observed with the untagged protein under our gel conditions.


( G ) atg1Δatg13Δ cells containing TAP ‐ tagged wild ‐ type Atg1 ( wt ) or the Atg1 kinase ‐ dead ( kd ) K54A mutant and wild ‐ type Atg13 , the Atg13 ‐ FV mutant or an empty plasmid were grown to mid log phase . Atg1 was immunoprecipitated and its autophosphorylation activity was measured by autoradiography in vitro .

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

(G) atg1Δatg13Δ cells containing TAP‐tagged wild‐type Atg1 (wt) or the Atg1 kinase‐dead (kd) K54A mutant and wild‐type Atg13, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase. Atg1 was immunoprecipitated and its autophosphorylation activity was measured by autoradiography in vitro.


( H ) pho8Δ60pho13Δatg13Δ cells expressing wild ‐ type Atg13 ( wt ) , the Atg13 ‐ FV mutant ( FV ) or an empty plasmid were grown to mid log phase and starved for 4 h in SD ‐ N medium . Pho8Δ60 ‐ specific alkaline phosphatase ( ALP ) activity ( nmol / min / mg ) was measured in three independent experiments as described in ' Materials and methods ' , and plotted as relative ALP activity with standard deviation ( s . d . ) compared to the wild ‐ type controls .

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

(H) pho8Δ60pho13Δatg13Δ cells expressing wild‐type Atg13 (wt), the Atg13‐FV mutant (FV) or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls.


( I ) atg13 cells expressing GFP ‐ Atg8 and containing wild ‐ type Atg13 , the Atg13 ‐ FV mutant or an empty plasmid were grown to mid log phase with and without starvation for 4 h in SD ‐ N . Processing of endogenous Ape1 and cleavage of GFP ‐ Atg8 was analysed by western blotting , and quantified by calculating the ratio of cleaved versus uncleaved Ape1 or GFP ‐ Atg8 , respectively . The asterisk marks a non ‐ specific band detected by the Atg13 antibody . Figure source data can be found with the Supplementary data .

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

(I) atg13 cells expressing GFP‐Atg8 and containing wild‐type Atg13, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase with and without starvation for 4 h in SD‐N. Processing of endogenous Ape1 and cleavage of GFP‐Atg8 was analysed by western blotting, and quantified by calculating the ratio of cleaved versus uncleaved Ape1 or GFP‐Atg8, respectively. The asterisk marks a non‐specific band detected by the Atg13 antibody. Figure source data can be found with the Supplementary data.


( B ) GST ‐ Atg8 or GST ‐ Ubiquitin ( Ub ) was purified from E . coli and bound to GSH beads . Wild ‐ type Atg1 ‐ TAP or the VE and EYE mutants were purified from yeast , incubated with the immobilized GST proteins and bound proteins analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GST‐Atg8 or GST‐Ubiquitin (Ub) was purified from E. coli and bound to GSH beads. Wild‐type Atg1‐TAP or the VE and EYE mutants were purified from yeast, incubated with the immobilized GST proteins and bound proteins analysed by western blotting.


( C ) ( C ) GST ‐ Atg8 and GST ‐ Ubiquitin were immobilized as described in ( B ) , and probed for their ability to bind Atg1 ‐ TAP isolated from wild ‐ type or atg13Δ cells .

O O O O O O O O O O O O O O O O O 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) (C) GST‐Atg8 and GST‐Ubiquitin were immobilized as described in (B), and probed for their ability to bind Atg1‐TAP isolated from wild‐type or atg13Δ cells.


( D ) atg1Δatg13Δ cells expressing as indicated wild ‐ type Atg1 ‐ TAP or the VE or EYE mutants , and wild ‐ type Atg13 or the Atg13 ‐ FV mutant were grown to mid log phase . Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) atg1Δatg13Δ cells expressing as indicated wild‐type Atg1‐TAP or the VE or EYE mutants, and wild‐type Atg13 or the Atg13‐FV mutant were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting.


( E ) GST , GST ‐ LC3B , GST ‐ GATE16 and GST ‐ GABARAP were purified from E . coli and bound to beads . HEK293 cell lysates prepared from cells expressing wild ‐ type ( wt ) HA ‐ ULK1 or the DFP mutant were incubated with the beads , and bound proteins analysed by western blotting .

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

(E) GST, GST‐LC3B, GST‐GATE16 and GST‐GABARAP were purified from E. coli and bound to beads. HEK293 cell lysates prepared from cells expressing wild‐type (wt) HA‐ULK1 or the DFP mutant were incubated with the beads, and bound proteins analysed by western blotting.


( F ) HEK293 cells were co ‐ transfected with GFP or GFP ‐ GABARAP along with either wild ‐ type HA ‐ ULK1 or the DFP mutant . GFP and GFP ‐ GABARAP were immobilized on GFP ‐ Trap resin and HA ‐ ULK1 binding was analysed by western blotting . Figure source data can be found with the Supplementary data .

(F) HEK293 cells were co‐transfected with GFP or GFP‐GABARAP along with either wild‐type HA‐ULK1 or the DFP mutant. GFP and GFP‐GABARAP were immobilized on GFP‐Trap resin and HA‐ULK1 binding was analysed by western blotting. Figure source data can be found with the Supplementary data.


( A ) atg1Δatg13Δ cells containing an empty control plasmid , wild ‐ type or the indicated Atg1 mutants , and Atg13 wild ‐ type , the Atg13 ‐ FVmutant or an empty plasmid were grown to mid log phase . Processing of endogenous Ape1 was analysed by western blotting , and quantified as described in the legend of Figure 1I . Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13 . The asterisk marks a non ‐ specific band detected by the Atg13 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) atg1Δatg13Δ cells containing an empty control plasmid, wild‐type or the indicated Atg1 mutants, and Atg13 wild‐type, the Atg13‐FVmutant or an empty plasmid were grown to mid log phase. Processing of endogenous Ape1 was analysed by western blotting, and quantified as described in the legend of Figure 1I. Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13. The asterisk marks a non‐specific band detected by the Atg13 antibody.


( B ) pho8Δ60pho13Δatg1Δ cells expressing wild ‐ type ( wt ) , kinase ‐ dead ( kd ) , Atg1 ‐ VE ( VE ) or ‐ EYE ( EYE ) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD ‐ N medium . Pho8Δ60 ‐ specific alkaline phosphatase ( ALP ) activity ( nmol / min / mg ) was measured in three independent experiments as described in ' Materials and methods ' , and plotted as relative ALP activity with standard deviation ( s . d . ) compared to the wild ‐ type controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) pho8Δ60pho13Δatg1Δ cells expressing wild‐type (wt), kinase‐dead (kd), Atg1‐VE (VE) or ‐EYE (EYE) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls.


( C ) Cells as in ( A ) expressing GFP ‐ Atg8 were grown to mid log phase with starvation for 4 h in SD ‐ N medium . The processing of GFP ‐ Atg8 was analysed and quantified as in Figure 1I .

O O O O O O O O O O O O O O O O O O O O O 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) Cells as in (A) expressing GFP‐Atg8 were grown to mid log phase with starvation for 4 h in SD‐N medium. The processing of GFP‐Atg8 was analysed and quantified as in Figure 1I.


( D ) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1 ‐ RFP and either GFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were examined by fluorescence microscopy . Bar = 5 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

(D) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1‐RFP and either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were examined by fluorescence microscopy. Bar=5 μm.


( E ) atg1Δ cells containing TAP ‐ tagged wild ‐ type or the indicated Atg1 ‐ mutants were grown to mid log phase . Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography . The kinase ‐ dead ( kd ) Atg1 ‐ mutant K54A serves as a negative control . Figure source data can be found with the Supplementary data .

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

(E) atg1Δ cells containing TAP‐tagged wild‐type or the indicated Atg1‐mutants were grown to mid log phase. Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography. The kinase‐dead (kd) Atg1‐mutant K54A serves as a negative control. Figure source data can be found with the Supplementary data.


( A ) Exponentially growing ypt7Δ cells were starved in SD ‐ N medium for 4 h , lysed and the extract separated into a cytoplasmic ( S ) and a 5000 g membrane pellet fraction . The pellet was treated with ( + ) or without ( − ) TX ‐ 100 , centrifuged and the supernatant ( S ) and pellet ( P ) fractions analysed by western blotting with anti ‐ Atg1 , anti ‐ Pep12 and anti ‐ Pgk1 antibodies .

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

(A) Exponentially growing ypt7Δ cells were starved in SD‐N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX‐100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti‐Atg1, anti‐Pep12 and anti‐Pgk1 antibodies.


( B ) Exponentially growing ypt7Δatg1Δ cells expressing wild ‐ type Atg1 or the Atg1 ‐ VE mutant protein were starved in SD ‐ N for 4 h , lysed and the extract separated into cytoplasmic ( S ) and membrane ( P ) fractions . The fractions were analysed by western blotting with anti ‐ Atg1 , anti ‐ Pgk1 and anti ‐ Ape1 antibodies , and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild ‐ type cells .

(B) Exponentially growing ypt7Δatg1Δ cells expressing wild‐type Atg1 or the Atg1‐VE mutant protein were starved in SD‐N for 4 h, lysed and the extract separated into cytoplasmic (S) and membrane (P) fractions. The fractions were analysed by western blotting with anti‐Atg1, anti‐Pgk1 and anti‐Ape1 antibodies, and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild‐type cells.


( C ) HEK293 cells were co ‐ transfected with GFP ‐ GABARAP and HA ‐ ULK1 , starved for 2 h , and subsequently immunostained for HA ( red ) and WIPI2 ( white ) . The arrows mark putative autophagosomes that stain positive for all three markers . 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

(C) HEK293 cells were co‐transfected with GFP‐GABARAP and HA‐ULK1, starved for 2 h, and subsequently immunostained for HA (red) and WIPI2 (white). The arrows mark putative autophagosomes that stain positive for all three markers. Bar=10 μm.


( D ) HEK293 cells stably expressing GFP ‐ LC3 were transfected with wild ‐ type HA ‐ ULK1 or the DFP mutant , and immunostained for HA and WIPI2 after 2 h starvation . HA ‐ ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells ( from one out of two experiments ) and plotted as the ratio of ULK1 to WIPI2 puncta . * * * Indicates a statistically significant P ‐ value of 0 . 0001 . One of the two independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells stably expressing GFP‐LC3 were transfected with wild‐type HA‐ULK1 or the DFP mutant, and immunostained for HA and WIPI2 after 2 h starvation. HA‐ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells (from one out of two experiments) and plotted as the ratio of ULK1 to WIPI2 puncta. ***Indicates a statistically significant P‐value of 0.0001. One of the two independent experiments is shown.


( E ) Quantification of HA ‐ ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild ‐ type HA ‐ ULK1 or the DFP mutant . Cells were treated as above , and the spot number counted using Imaris software . A representative immunofluorescence image is shown in Supplementary Figure S3F . * * * and * indicate P ‐ values of 0 . 0001 or 0 . 0158 , respectively . Figure source data can be found with the Supplementary data .

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

(E) Quantification of HA‐ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild‐type HA‐ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P‐values of 0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.


( A ) atg1Δ cells containing either wild ‐ type Atg1 or the kinase ‐ dead Atg1 ‐ T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD ‐ N . Samples were taken at indicated time points and GFP cleavage was analysed by western blotting . Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) atg1Δ cells containing either wild‐type Atg1 or the kinase‐dead Atg1‐T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD‐N. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux.


( B ) atg1Δ cells containing either GFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were grown and analysed as in ( A ) .

O O O O O O 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) atg1Δ cells containing either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown and analysed as in (A).


( C ) atg1Δpep4Δ cells containing YFP ‐ tagged wild ‐ type Atg1 or the Atg1 ‐ VE mutant were grown to log phase and starved in SD ‐ N . Cells were monitored by time ‐ lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in ' Materials and methods ' . Data are plotted as the mean ± s . e . m . from at least 90 quantified cells . Additional images are shown in Supplementary Figure S3H . Bar = 5 μm .

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(C) atg1Δpep4Δ cells containing YFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown to log phase and starved in SD‐N. Cells were monitored by time‐lapse microscopy and the vacuolar accumulation of Atg1 was quantified as described in 'Materials and methods'. Data are plotted as the mean±s.e.m. from at least 90 quantified cells. Additional images are shown in Supplementary Figure S3H. Bar=5 μm.


( D ) atg1Δatg13Δ cells expressing GFP ‐ tagged wild ‐ type Atg13 , and either wild ‐ type Atg1 or the Atg1 ‐ VE mutant ( left panel ) and wild ‐ type cells containing Atg13 ‐ GFP and either wild ‐ type Atg13 or Atg13 ‐ FV were grown to mid log phase followed by starvation for 6 h in SD ‐ N media . GFP cleavage was analysed by western blotting . Figure source data can be found with the Supplementary data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) atg1Δatg13Δ cells expressing GFP‐tagged wild‐type Atg13, and either wild‐type Atg1 or the Atg1‐VE mutant (left panel) and wild‐type cells containing Atg13‐GFP and either wild‐type Atg13 or Atg13‐FV were grown to mid log phase followed by starvation for 6 h in SD‐N media. GFP cleavage was analysed by western blotting. Figure source data can be found with the Supplementary data.


A , B Representative images of OG immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of OG immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125


A , B Representative images o O - GlcNAc ( B ) immunohistochemistry on paraffin - embedded colon sections from control , UC and CD patients ( n = 4 ) . Intensities of staining were scored on the right . Scale bars = 50 μm . ( one - way ANOVA , OGT : Healthy vs UC P = 0 . 0002 , Healthy vs CD P = 0 . 003 ; O - GlcNAc : Healthy vs UC P = 0 . 0365 , Healthy vs CD P = 0 .

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A, B Representative images o O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (one-way ANOVA, OGT: Healthy vs UC P=0.0002, Healthy vs CD P=0.003; O-GlcNAc: Healthy vs UC P=0.0365, Healthy vs CD P=0.0125


C Levels of intestinal epithelial OGT and O - GlcNAcylation in a second cohort of control , UC , and CD patients . ( one - way ANOVA , OGT : Control n = 10 , UC n = 9 , CD n = 6 ; Control vs UC P = 0 . 035 , Control vs CD P = 0 . 003 ; O - GlcNAc : Control n = 10 , UC n = 8 , CD n = 8 ; Control vs UC P = 0 . 0027 , Control vs CD P = 0 . 008

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C Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (one-way ANOVA, OGT: Control n=10, UC n=9, CD n=6; Control vs UC P=0.035, Control vs CD P=0.003; O-GlcNAc: Control n=10, UC n=8, CD n=8; Control vs UC P=0.0027, Control vs CD P=0.008


D , E Regression plots of average immune - staining scores of OGT and O - GlcNAcylation against histological scores in UC ( D ) and CD ( E ) ( D : OGT n = 19 , O - GlcNAc n = 18 ; E : OGT n = 15 , O - GlcNAc n = 16 )

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D, E Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n=19, O-GlcNAc n=18; E: OGT n=15, O-GlcNAc n=16)


A Body weight of male wildtype and Vil - Ogt KO mice at 6 weeks of age ( n = 6 , P = 0 . 000015 )

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A Body weight of male wildtype and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015)


B Incidence of rectal prolapse in male wildtype and Vil - Ogt KO mice ( WT n = 17 , KO n = 9 , P < 0 . 0001 )

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B Incidence of rectal prolapse in male wildtype and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001)


C Representative colonoscopy images of male wildtype and Vil - Ogt KO mice

C Representative colonoscopy images of male wildtype and Vil-Ogt KO mice


D Body weight of female wildtype and Vil - Ogt KO mice at 9 weeks of age ( WT n = 11 , heterozygous KO n = 4 , homozygous KO n = 5 , P < 0 . 0001 )

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D Body weight of female wildtype and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001)


E Incidence of rectal prolapse in female wildtype and Vil - Ogt KO mice ( WT n = 9 , heterozygous KO n = 8 , homozygous KO n = 6 , P = 0 . 0439 )

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E Incidence of rectal prolapse in female wildtype and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439)


F H & E staining of duodenum , jejunum , ileum , colon , and rectum of 10 - week - old male wildtype and Vil - Ogt KO mice . Scale bars = 100 μm

F H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wildtype and Vil-Ogt KO mice. Scale bars = 100 μm


A Numbers of Paneth cells in the ileum of wildtype and Vil - Ogt KO mice ( n = 6 , P = 0 . 0048 )

A Numbers of Paneth cells in the ileum of wildtype and Vil-Ogt KO mice (n = 6, P = 0.0048)


B Representative electron microscopic images of Paneth cells . Scale bars = 1 μm

B Representative electron microscopic images of Paneth cells. Scale bars = 1 μm


C Expression of AMP genes in ileum tissues of wildtype and Vil - Ogt KO mice ( n = 6 , Pan Defesin P = 0 . 031 , Defa3 P = 0 . 049 , Defa4 P = 0 . 013 , Defa5 P = 0 . 023 , Defa6 P = 0 . 034 , Ang4 P = 0 . 031 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Expression of AMP genes in ileum tissues of wildtype and Vil-Ogt KO mice (n = 6, Pan Defesin P = 0.031, Defa3 P = 0.049, Defa4 P = 0.013, Defa5 P = 0.023, Defa6 P = 0.034, Ang4 P = 0.031)


D Immunoblotting of LC3 in IEC cells isolated from WT and Vil - Ogt KO mice

D Immunoblotting of LC3 in IEC cells isolated from WT and Vil-Ogt KO mice


E Representative images of O - GlcNAc staining in Paneth cells of male wildtype and Defa6 - Ogt KO mice . Stars indicate lysozyme - positive Paneth cells

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E Representative images of O-GlcNAc staining in Paneth cells of male wildtype and Defa6-Ogt KO mice. Stars indicate lysozyme-positive Paneth cells


F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 , P = 0 . 024 ) . Arrow indicate Paneth cell . Scale bars = 10 μm

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F Morphology and numbers of Paneth cells in the ileum of wildtype and Defa6-Ogt KO mice (n = 5, P = 0.024). Arrow indicate Paneth cell. Scale bars = 10 μm


G Expression of AMP genes in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 Pan Defesin P = 0 . 0291 , Defa3 P = 0 . 0462 )

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G Expression of AMP genes in the ileum of wildtype and Defa6-Ogt KO mice (n = 5 Pan Defesin P = 0.0291, Defa3 P = 0.0462)


H Representative images of cultured organoid on Day 3


I Expression of inflammatory markers in the ileum of wildtype and Defa6 - Ogt KO mice ( n = 5 )

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I Expression of inflammatory markers in the ileum of wildtype and Defa6-Ogt KO mice (n = 5)


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes in body weight ( J ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes in body weight (J) are shown (n = 4-5). (L: P = 0.049


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis . Daily changes i colitis scores ( K are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis. Daily changes i colitis scores (K are shown (n = 4-5). (L: P = 0.049


J - L Antibiotic - treated wildtype and Defa6 - Ogt KO male mice were transplanted with fecal microbiota from Vil - Ogt KO mice and then induced with DSS for colitis RT - qPCR of inflammatory genes in the ileum ( L ) are shown ( n = 4 - 5 ) . ( L : P = 0 .

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J-L Antibiotic-treated wildtype and Defa6-Ogt KO male mice were transplanted with fecal microbiota from Vil-Ogt KO mice and then induced with DSS for colitis RT-qPCR of inflammatory genes in the ileum (L) are shown (n = 4-5). (L: P = 0.049


A Expression of phosphorylated and total STAT3 and NF - κB p65 in the colon of wildtype and Vil - Ogt KO mice

A Expression of phosphorylated and total STAT3 and NF-κB p65 in the colon of wildtype and Vil-Ogt KO mice


B Ileum and colon IECs from 10 - week - old wildtype and Vil - Ogt KO mice were isolated for RNA - sequencing analyses . The heatmaps of the sample - to - sample distances are shown

B Ileum and colon IECs from 10-week-old wildtype and Vil-Ogt KO mice were isolated for RNA-sequencing analyses. The heatmaps of the sample-to-sample distances are shown


C Volcano plots of gene expression changes . The x - axis specifies the fold - changes ( FC ) and the y - axis specifies the negative logarithm to the base 10 of the p - values adjusted for multiple testing with the Benjamini - Hochberg procedure . FC > 2 and p < 0 . 05 genes are shown in solid dots

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C Volcano plots of gene expression changes. The x-axis specifies the fold-changes (FC) and the y-axis specifies the negative logarithm to the base 10 of the p-values adjusted for multiple testing with the Benjamini-Hochberg procedure. FC > 2 and p < 0.05 genes are shown in solid dots


F Levels of STAT1 protein in IECs determined by immunoblotting and O - GlcNAc modification of STAT1 determined by immunoprecipitation using the O - GlcNAc antibody followed by Immunoblotting

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F Levels of STAT1 protein in IECs determined by immunoblotting and O-GlcNAc modification of STAT1 determined by immunoprecipitation using the O-GlcNAc antibody followed by Immunoblotting


G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 and global O - GlcNAcylatio were

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G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 and global O-GlcNAcylatio were determined


G , H Caco - 2 cells were treated with 100 uM Ac45S - GlcNAc for 24 hours , levels of STAT1 an STAT1 O - GlcNAcylation ( H ) were

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G, H Caco-2 cells were treated with 100 uM Ac45S-GlcNAc for 24 hours, levels of STAT1 an STAT1 O-GlcNAcylation (H) were determined


I siRNAs against scrambled sequence or human OGT were transfected in 293T cells and STAT1 levels were determined 48 hours later

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J The pGAS - luc Cis - Reporter plasmid was co - transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 ( n = 4 , P = 0 . 0003 )

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

J The pGAS-luc Cis-Reporter plasmid was co-transfected with siRNAs in 293T cells for luciferase assay to determine the transcriptional activity of STAT1 (n = 4, P=0.0003)


A , B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS . Daily changes in body weight ( A ) and colitis score ( B ) ( n = 5 ) . ( B : day6 P = 0 . 0171 , day7 P = 0 . 0445

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A, B Wildtype mice were treated with H2O or TMG for 2 weeks before the induction of colitis by DSS. Daily changes in body weight (A) and colitis score (B) (n= 5). (B: day6 P = 0.0171, day7 P= 0.0445


C Intestinal barrier functional assays measuring serum FITC - dextran before DSS treatment ( n = 5 , P = 0 . 0117 )

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C Intestinal barrier functional assays measuring serum FITC-dextran before DSS treatment (n = 5, P = 0.0117)


D - G Albumin levels in feces ( D on day 4 of recovery ( n = 5 )

D-G Albumin levels in feces (D on day 4 of recovery (n = 5)


colon length ( E ) on day 4 of recovery ( n = 5 )

colon length (E) on day 4 of recovery (n = 5)


H & E staining of colon tissues ( F on day 4 of recovery ( n = 5 ) . Scale bars = 100

H&E staining of colon tissues (F on day 4 of recovery (n = 5). Scale bars = 100 μm


RT - qPCR of inflammatory genes in the colon ( G ) on day 4 of recovery ( n = 5 ) . Scale bars = 100 μm . ( E : P = 0 . 0474 ; G : Tnfa P = 0 . 0190 , Il1b P = 0 .

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

RT-qPCR of inflammatory genes in the colon (G) on day 4 of recovery (n = 5). Scale bars = 100 μm. (E: P = 0.0474; G: Tnfa P = 0.0190, Il1b P = 0.0301


( A ) Quantitative RT - PCR analysis of M . tb RNA in EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . ND , not detected . Scale bar , 20 μM . Data shown in ( A ) are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( 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

(A) Quantitative RT-PCR analysis of M.tb RNA in EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. ND, not detected. Scale bar, 20 μM. Data shown in (A) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates).


Quantitative RT - PCR analysis for IFN - β mRNA ( B ) levels in wild type BMMs 5 and 24 hr post - treatment with EVs respectively ( 1 μg / ml equals approximately 3 x 108 vesicles / ml ) . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-PCR analysis for IFN-β mRNA (B) levels in wild type BMMs 5 and 24 hr post-treatment with EVs respectively (1 μg/ml equals approximately 3 x 108 vesicles/ml). Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


IFN - β protein ( C ) levels in wild type BMMs 5 and 24 hr post - treatment with EVs respectively ( 1 μg / ml equals approximately 3 x 108 vesicles / ml ) . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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

IFN-β protein (C) levels in wild type BMMs 5 and 24 hr post-treatment with EVs respectively (1 μg/ml equals approximately 3 x 108 vesicles/ml). Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( D ) IFN - β mRNA level was quantified in wild type BMMs treated with EVs at a concentration of 10μg / ml for the times indicated . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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(D) IFN-β mRNA level was quantified in wild type BMMs treated with EVs at a concentration of 10μg/ml for the times indicated. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


Quantitative RT - PCR analysis for IFN - β mRNA ( E ) levels in either WT , MAVS - and / or RIG - I - knockdown BMMs treated for 4 hr ( E ) with 10µg / ml EVs from uninfected or M . tb - infected macrophages . ( Control ) negative control siRNA . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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Quantitative RT-PCR analysis for IFN-β mRNA (E) levels in either WT, MAVS- and/or RIG-I-knockdown BMMs treated for 4 hr (E) with 10µg/ml EVs from uninfected or M.tb-infected macrophages. (Control) negative control siRNA. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


IFN - β protein ( F ) levels in either WT , MAVS - and / or RIG - I - knockdown BMMs treated for 24 hr ( F ) with 10µg / ml EVs from uninfected or M . tb - infected macrophages . ( Control ) negative control siRNA . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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

IFN-β protein (F) levels in either WT, MAVS- and/or RIG-I-knockdown BMMs treated for 24 hr (F) with 10µg/ml EVs from uninfected or M.tb-infected macrophages. (Control) negative control siRNA. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( G ) Similar to above except using WT and Mavs - / - BMMs . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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(G) Similar to above except using WT and Mavs -/- BMMs. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( H ) Similar to above except using WT and Mavs - / - BMMs . Scale bar , 20 μM . Data shown in ( H ) are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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(H) Similar to above except using WT and Mavs -/- BMMs. Scale bar, 20 μM. Data shown in (H) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( J ) Top chamber : WT BMMs ; Bottom chamber : WT or Mavs - / - BMMs . Data shown in ( J ) are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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(J) Top chamber: WT BMMs; Bottom chamber: WT or Mavs -/- BMMs. Data shown in (J) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( K ) Western blot analysis for phospho - TBK1 ( Ser172 ) in WT and MAVS - knockdown BMMs treated for 4 hr with EVs from uninfected or M . tb - infected macrophages . β - actin served as a loading control . Densitometry of the western blots for ( K ) are shown .

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(K) Western blot analysis for phospho-TBK1 (Ser172) in WT and MAVS-knockdown BMMs treated for 4 hr with EVs from uninfected or M.tb-infected macrophages. β-actin served as a loading control. Densitometry of the western blots for (K) are shown.


( L ) Western blot analysis of IRF3 under the conditions described above for TBK1 . Histone H3 ( H3 ) and β - actin were used as loading controls for nuclear fraction and whole - cell lysate ( WCL ) respectively . Densitometry of the western blots are shown .

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(L) Western blot analysis of IRF3 under the conditions described above for TBK1. Histone H3 (H3) and β-actin were used as loading controls for nuclear fraction and whole-cell lysate (WCL) respectively. Densitometry of the western blots are shown.


( M ) Fluorescence microscopy analysis for nuclear translocation of IRF3 in wild type BMMs untreated ( Mock ) or treated with EVs from uninfected or M . tb - infected macrophages . Scale bar , 20 μM .

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(M) Fluorescence microscopy analysis for nuclear translocation of IRF3 in wild type BMMs untreated (Mock) or treated with EVs from uninfected or M.tb-infected macrophages. Scale bar, 20 μM.


( N ) IRF3 nuclear translocation was analyzed in RIG - I - or MAVS - knockdown BMMs . Scale bar , 20 μM .

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(N) IRF3 nuclear translocation was analyzed in RIG-I- or MAVS-knockdown BMMs. Scale bar, 20 μM.


( C ) Yield of purified EVs from BMMs infected with various M . tb CDC 1551 strains based on Nanosight analysis . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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(C) Yield of purified EVs from BMMs infected with various M.tb CDC 1551 strains based on Nanosight analysis. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


( B ) EVs from cells infected with WT , ∆ esxA or esxA - complemented ( ∆ esxA - C ) M . tb Erdman strains .

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(B) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains.


( D ) EVs from cells infected with WT , ∆ esxA or esxA - complemented ( ∆ esxA - C ) M . tb Erdman strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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(D) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


qRT - PCR analysis for M . tb RNA in EVs from BMMs infected with various M . tb CDC 1551 strains ( E ) ND , not detected . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb CDC 1551 strains (E) ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


qRT - PCR analysis for M . tb RNA in EVs from BMMs infected with various M . tb Erdman strains ( F ) . ND , not detected . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb Erdman strains (F). ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


qRT - PCR analysis for IFN - β mRNA ( G ) in WT BMMs treated with EVs for 4 hr and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb CDC 1551 strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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qRT-PCR analysis for IFN-β mRNA (G) in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


IFN - β protein ( H ) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb CDC 1551 strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

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IFN-β protein (H) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


( I ) ELISA analysis for IFN - β secreted by BMMs treated with EVs plus liposomes containing M . tb RNA . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

(I) ELISA analysis for IFN-β secreted by BMMs treated with EVs plus liposomes containing M.tb RNA. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


qRT - PCR analysis for IFN - β mRNA ( J ) in WT BMMs treated with EVs for 4 and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb Erdman strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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qRT-PCR analysis for IFN-β mRNA (J) in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


IFN - β protein ( K ) levels in WT BMMs treated with EVs for 4 and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb Erdman strains . Data shown in K ) are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

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IFN-β protein (K) levels in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown in K) are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


M . tb CFU in WT mouse BMMs pre - treated with EVs minus co - treatment with IFN - ɣ . BMMs were treated with EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) cells for 0 , 24 , 48 and 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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M.tb CFU in WT mouse BMMs pre-treated with EVs minus co-treatment with IFN-ɣ. BMMs were treated with EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) cells for 0, 24, 48 and 72 hr following a 1 hr M.tb infection. Mock, no EV treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


M . tb CFU in WT mouse BMMs pre - treated with EVs plus ( B ) co - treatment with IFN - ɣ . BMMs were treated with EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) cells for 0 , 24 , 48 and 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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M.tb CFU in WT mouse BMMs pre-treated with EVs plus (B) co-treatment with IFN-ɣ. BMMs were treated with EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) cells for 0, 24, 48 and 72 hr following a 1 hr M.tb infection. Mock, no EV treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy analysis for colocalization of M . tb ( GFP ) with lysosome marker Lamp - 1 in WT mouse BMMs . Cells were pre - treated for 5 hr with EVs from uninfected or M . tb - infected macrophages plus IFN - γ , and then infected with GFP - expressing M . tb for 24 hr prior to immunostaining . quantitative analysis of M . tb colocalization with Lamp - 1 in WT mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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Immunofluorescence microscopy analysis for colocalization of M.tb (GFP) with lysosome marker Lamp-1 in WT mouse BMMs. Cells were pre-treated for 5 hr with EVs from uninfected or M.tb-infected macrophages plus IFN-γ, and then infected with GFP-expressing M.tb for 24 hr prior to immunostaining. quantitative analysis of M.tb colocalization with Lamp-1 in WT mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy analysis for colocalization of M . tb ( GFP ) with lysosome marker Lamp - 1 in Mavs - / - ( E and F ) mouse BMMs . Cells were pre - treated for 5 hr with EVs from uninfected or M . tb - infected macrophages plus IFN - γ , and then infected with GFP - expressing M . tb for 24 hr prior to immunostaining . quantitative analysis of M . tb colocalization with Lamp - 1 in Mavs - / - mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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Immunofluorescence microscopy analysis for colocalization of M.tb (GFP) with lysosome marker Lamp-1 in Mavs-/- (E and F) mouse BMMs. Cells were pre-treated for 5 hr with EVs from uninfected or M.tb-infected macrophages plus IFN-γ, and then infected with GFP-expressing M.tb for 24 hr prior to immunostaining. quantitative analysis of M.tb colocalization with Lamp-1 in Mavs-/- mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy as described above except using control siRNA - treated ( G and H ) BMMs . quantitative analysis of M . tb colocalization with Lamp - 1 in control siRNA - treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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Immunofluorescence microscopy as described above except using control siRNA-treated (G and H) BMMs. quantitative analysis of M.tb colocalization with Lamp-1 in control siRNA-treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Immunofluorescence microscopy as described above except using RIG - I siRNA - treated ( I and J ) BMMs . quantitative analysis of M . tb colocalization with Lamp - 1 in RIG - I siRNA - treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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Immunofluorescence microscopy as described above except using RIG-I siRNA-treated (I and J) BMMs. quantitative analysis of M.tb colocalization with Lamp-1 in RIG-I siRNA-treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


M . tb CFU in infected Mavs - / - ( K ) mouse BMMs pre - treated with IFN - ɣ plus EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . CFU was determined immediately after the 1 hr infection or 24 and 72 hr post - infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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M.tb CFU in infected Mavs-/- (K) mouse BMMs pre-treated with IFN-ɣ plus EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. CFU was determined immediately after the 1 hr infection or 24 and 72 hr post-infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


M . tb CFU in control siRNA - treated ( L ) or RIG - I siRNA - treated ( M ) mouse BMMs pre - treated with IFN - ɣ plus EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . CFU was determined immediately after the 1 hr infection or 24 and 72 hr post - infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

M.tb CFU in control siRNA-treated (L) or RIG-I siRNA-treated (M) mouse BMMs pre-treated with IFN-ɣ plus EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. CFU was determined immediately after the 1 hr infection or 24 and 72 hr post-infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.