E : Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Right panel shows the quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

E: Cell lysates from control and ELDR depleted Cal27 and JHU029 cells were subjected to Western blot analysis for ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Right panel shows the quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


F : Control , ELDR overexpressed and ELDR - depleted Cal27 cells were probed with an antibody against ILF3 ( red ) and DAPI ( blue ) . Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3 . Arrows indicate cytoplasmic expression . Magnifications 60X , scale bar 20 μ .

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

F: Control, ELDR overexpressed and ELDR-depleted Cal27 cells were probed with an antibody against ILF3 (red) and DAPI (blue). Representative confocal microscopic images show nuclear and cytoplasmic expression of ILF3. Arrows indicate cytoplasmic expression. Magnifications 60X, scale bar 20 μ.


A : Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates . Relative mRNA expression of EGFR was analyzed by qRT - PCR . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

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

A: Cal27 and JHU022 cell lysates were immunoprecipitated with control or ILF3 antibodies and RNA was isolated from the precipitates. Relative mRNA expression of EGFR was analyzed by qRT-PCR. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


B : Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . n = 3 biological replicates .

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

B: Cal27 and JHU029 cells were transfected with control or ILF3 siRNA and after 48hr cell lysates were subjected to Western blot analysis for EGFR and ILF3 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. n = 3 biological replicates.


C : Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT - PCR . 18S was used as internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O 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: Cal27 and JHU029 cell lysates were transfected with either siELDR or siILF3 and relative mRNA expression of Cyclin E1 was analyzed by qRT-PCR. 18S was used as internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


D : Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody . The membrane was reprobed with an antibody against Actin as an internal control . Bottom panels show quantitation . n = 4 biological replicates . Data are represented as the mean ± SD ,

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

D: Control or ELDR depleted Cal27 and JHU029 cell lysates using two different siRNAs to ELDR were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Bottom panels show quantitation. n = 4 biological replicates. Data are represented as the mean ± SD,


E : Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody . The membrane was reprobed with an antibody against Actin as internal control . Bottom panel shows quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

E: Control or ELDR overexpressed NOK lysates were subjected to Western blot analysis for Cyclin E1 using specific antibody. The membrane was reprobed with an antibody against Actin as internal control. Bottom panel shows quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


F : Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Bottom panels show quantitation . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

F: Control or ILF3depleted Cal27 and JHU029 cell lysates were subjected to Western blot analysis for Cyclin E1 using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD,


G : Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho - RB and total RB using specific antibodies . The membrane was reprobed with an antibody against Actin as internal control . Bottom panels show quantitation . n = 3 biological replicates . Data are represented as the mean ±

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

G: Cal27 and JHU029 cells were transfected with either siELDR or siILF3 and after 48hr cell lysates were subjected to Western blot analysis for phospho-RB and total RB using specific antibodies. The membrane was reprobed with an antibody against Actin as internal control. Bottom panels show quantitation. n = 3 biological replicates. Data are represented as the mean ± SD


H : Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested , fixed and stained with propidium iodide . DNA content was analyzed by flow cytometry . Results are represented cell population in G1 , S , and G2 / M phases of the cell cycle . Right panels show percentage of cell population in different phases of cell cycle . n = 4 biological replicates . Data are represented as the mean ± SD ,

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 O O O O O O

H: Control or ELDR knockdown synchronized Cal27 and JHU029 cells were harvested, fixed and stained with propidium iodide. DNA content was analyzed by flow cytometry. Results are represented cell population in G1, S, and G2/M phases of the cell cycle. Right panels show percentage of cell population in different phases of cell cycle. n = 4 biological replicates. Data are represented as the mean ± SD,


A : Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR - 7 was analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

A: Cal27 and JHU029 cells were transfected with control or ELDR siRNA and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


B : Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR - 7 was analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates . Data are represented as the mean ± SD ,

B: Cal27 and JHU029 cells were transfected with control or ELDR plasmid and relative expression of miR-7 was analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates. Data are represented as the mean ± SD,


C : Cal27 and JHU029 cells were transfected with control or mimic miR - 7 and relative expression of ELDR was analyzed by qRT - PCR . 18S rRNA was used as an internal control . n = 2 biological replicates ; n = 3 technical replicates . Data are represented as the mean ± SD ,

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

C: Cal27 and JHU029 cells were transfected with control or mimic miR-7 and relative expression of ELDR was analyzed by qRT-PCR. 18S rRNA was used as an internal control. n = 2 biological replicates; n= 3 technical replicates. Data are represented as the mean ± SD,


D : Cal27 and JHU029 cells were transfected with miR - 7 mimic , ELDR plasmid or both miR - 7 and ELDR plasmid . Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody . The membrane was reprobed with an antibody against Actin as an internal control . Representative bands were cropped from same gel . Right panels show quantitative representation of Western blot band intensities . n = 3 biological replicates . Data are represented as the mean ± SD ,

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

D: Cal27 and JHU029 cells were transfected with miR-7 mimic, ELDR plasmid or both miR-7 and ELDR plasmid. Cell lysates were analyzed by Western blot analysis for EGFR expression using specific antibody. The membrane was reprobed with an antibody against Actin as an internal control. Representative bands were cropped from same gel. Right panels show quantitative representation of Western blot band intensities. n = 3 biological replicates. Data are represented as the mean ± SD,


E : Relative expression of miR - 7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT - PCR . U47 was used as an internal control . n = 3 biological replicates and 3 technical replicates . Data are represented as the mean ± SD ,

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

E: Relative expression of miR-7 in Cal27 and JHU029 cells compared to NOK cell line analyzed by qRT-PCR. U47 was used as an internal control. n = 3 biological replicates and 3 technical replicates. Data are represented as the mean ± SD,


A : Body weight was measured in control and siELDR treated mice . n = 5 animal per

A: Body weight was measured in control and siELDR treated mice. n=5 animal per group


B : Representative images of tumors in control and treatment groups .

B: Representative images of tumors in control and treatment groups.


C : Tumors were measured using a slide caliper and tumor volumes were calculated . Arrow indicates starting point siELDR treatment . n = 5 animal per group and tumor volume presented as the mean ±

O O O O O O O O O O O O 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: Tumors were measured using a slide caliper and tumor volumes were calculated. Arrow indicates starting point siELDR treatment. n=5 animal per group and tumor volume presented as the mean ± SD


D : ELDR knockdown was examined by analyzing expression of ELDR in treated tumors compared to the control tumors . 18S rRNA gene was used as internal control . n = 5 animal per group and 3 technical replicates from each tumor .

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

D: ELDR knockdown was examined by analyzing expression of ELDR in treated tumors compared to the control tumors. 18S rRNA gene was used as internal control. n=5 animal per group and 3 technical replicates from each tumor.


E : Control or siELDR treated tumor lysates were subjected to Western blot analysis for EGFR and Cyclin E1expression using specific antibodies and representative bands are shown . The blot was reprobed with an antibody against Actin for normalization . Right panels show quantitation . n = 3 biological replicates from each

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

E: Control or siELDR treated tumor lysates were subjected to Western blot analysis for EGFR and Cyclin E1expression using specific antibodies and representative bands are shown. The blot was reprobed with an antibody against Actin for normalization. Right panels show quantitation. n= 3 biological replicates from each tumor


F : OSCC - PDX tissue was implanted subcutaneously into the flank of NSG mice . When the tumor volume reached > 70 mm3 , mice were divided into two groups . 10µg of siELDR or control oligoes were injected intratumorally as described above . Body weight was measured in control and siELDR treated mice . n = 5 animal per

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: OSCC-PDX tissue was implanted subcutaneously into the flank of NSG mice. When the tumor volume reached >70 mm3, mice were divided into two groups. 10µg of siELDR or control oligoes were injected intratumorally as described above. Body weight was measured in control and siELDR treated mice. n=5 animal per group


G : Representative images of OSCC - PDX tumors in control and treatment groups .

G: Representative images of OSCC-PDX tumors in control and treatment groups.


H : PDX tumors were measured using a slide caliper and tumor volumes were calculated . n = 5 animal per group and tumor volume presented as the mean ±

O O O O O O 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: PDX tumors were measured using a slide caliper and tumor volumes were calculated. n=5 animal per group and tumor volume presented as the mean ± SD


I : ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC - PDX tumors compared to the control tumors . 18S gene was used as internal control . n = 5 animal per group and 3 technical replicates presented as the mean ±

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

I: ELDR knockdown was examined by analyzing expression of ELDR in treated OSCC-PDX tumors compared to the control tumors. 18S gene was used as internal control. n=5 animal per group and 3 technical replicates presented as the mean ± SD


( A - C ) The developing tumor vasculature was imaged over time in an abdominal window chamber model with two - photon microscopy with ( A ) GFP - labeled tumor cells , ( B ) TdTomato labeled TECs in cyan and ( C ) perfusion . ( D ) Top : a representative image of MC38 tumor vasculature , bottom : segmentation with skeletonization . ( E - N ) The following parameters were quantified from the segmented image each day : ( E ) vessel tortuosity , ( F ) directional coherence , ( G ) perfusion , ( H ) number of sprouts , ( I ) fraction of branches < 80 µm , ( J ) fraction of branches > 400 µm , ( K ) length to diameter ratio , ( L ) nodes per mm2 , ( M ) vessel length , and ( N ) vessel diameter .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) The developing tumor vasculature was imaged over time in an abdominal window chamber model with two-photon microscopy with (A) GFP-labeled tumor cells, (B) TdTomato labeled TECs in cyan and (C) perfusion. (D) Top: a representative image of MC38 tumor vasculature, bottom: segmentation with skeletonization. (E-N) The following parameters were quantified from the segmented image each day: (E) vessel tortuosity, (F) directional coherence, (G) perfusion, (H) number of sprouts,(I) fraction of branches <80 µm, (J) fraction of branches >400 µm, (K) length to diameter ratio, (L) nodes per mm2, (M) vessel length, and (N) vessel diameter.


The developing MC38 tumor vasculature was imaged in an abdominal window chamber model with two - photon microscopy after IR at the indicated times . ( A ) ( top ) Representative two - photon images of a control MC38 tumor vasculature ( cyan ) and perfusion ( red ) at day 2 and 4 . ( Bottom ) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature ( cyan ) and perfusion ( red ) . ( B - C ) Single dose 15 Gy IR ( B ) and 5 × 3 Gy ( C ) fractionated IR treated MC38 tumor vasculature ( top ) at day 2 and day 4 . ( Bottom ) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature ( cyan ) and perfusion ( red ) .

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

The developing MC38 tumor vasculature was imaged in an abdominal window chamber model with two-photon microscopy after IR at the indicated times. (A) (top) Representative two-photon images of a control MC38 tumor vasculature (cyan) and perfusion (red) at day 2 and 4. (Bottom) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature (cyan) and perfusion (red). (B-C) Single dose 15 Gy IR (B) and 5×3 Gy (C) fractionated IR treated MC38 tumor vasculature (top) at day 2 and day 4. (Bottom) zoom of the same tumor region from day 0 to day 4 of the tumor vasculature (cyan) and perfusion (red).


( E - L ) Quantified vascular parameters , for each day of imaging the following parameters were quantified from segmented images and normalized to day one if indicated : ( E ) normalized node density , ( F ) normalized tortuosity , ( G ) normalized branch length < 80μm , ( H ) normalized branch length > 400μm , ( I ) vessel branch length , ( J ) vessel diameter , ( K ) normalized length to diameter ratio , ( L ) normalized perfused vessels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) Quantified vascular parameters, for each day of imaging the following parameters were quantified from segmented images and normalized to day one if indicated: (E) normalized node density , (F) normalized tortuosity, (G) normalized branch length < 80μm, (H) normalized branch length > 400μm, (I) vessel branch length, (J) vessel diameter, (K) normalized length to diameter ratio, (L) normalized perfused vessels.


( M - N ) Subcutaneous tumor growth measurements of MC38 ( M ) and B16F10 ( N ) tumors .

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

(M-N) Subcutaneous tumor growth measurements of MC38 (M) and B16F10 (N) tumors.


( A - B ) Representative immunofluorescent micrographs of control and treated tumor vasculature from MC38 tumors . Tumor vessels ( anti - CD31 ) and apoptosis ( anti - cleaved caspase 3 ) in ( A ) control and ( B ) 48 hours after 15 Gy IR . Arrows indicate apoptotic TEC . ( C ) quantification of apoptotic vessels after single dose 15 Gy IR from immunofluorescent images of whole MC38 tumor section ( n = 6 - 8 biological replicates per group ) . ( D ) Quantification of apoptotic vessels after fractionated 5 × 3 Gy IR from immunofluorescent images of whole MC38 tumor sections ( n = 6 - 8 biological replicates per group ) . ( E ) Quantification of apoptosis in TECs from B16F10 tumor sections ( n = 4 - 6 biological replicates per group ) . ( F ) Quantification of apoptotic vessels according to size on thin whole MC38 tumor sections ( Ctrl n = 4 and treatment n = 7 biological replicates ) . ( G ) Quantification of apoptotic vessels according to size in B16F10 tumor sections ( Ctrl n = 4 and treatment n = 10 biological replicates ) . ( H - I ) Area ( H ) and diameter ( I ) of apoptotic tumor vessels vs all tumor vessels from segmented thick 80 μm MC38 tumor sections ( n = 4 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescent micrographs of control and treated tumor vasculature from MC38 tumors. Tumor vessels (anti-CD31) and apoptosis (anti-cleaved caspase 3) in (A) control and (B) 48 hours after 15 Gy IR. Arrows indicate apoptotic TEC. (C) quantification of apoptotic vessels after single dose 15 Gy IR from immunofluorescent images of whole MC38 tumor section (n= 6-8 biological replicates per group). (D) Quantification of apoptotic vessels after fractionated 5×3 Gy IR from immunofluorescent images of whole MC38 tumor sections (n= 6-8 biological replicates per group). (E) Quantification of apoptosis in TECs from B16F10 tumor sections (n= 4-6 biological replicates per group). (F) Quantification of apoptotic vessels according to size on thin whole MC38 tumor sections (Ctrl n=4 and treatment n=7 biological replicates). (G) Quantification of apoptotic vessels according to size in B16F10 tumor sections (Ctrl n=4 and treatment n= 10 biological replicates). (H-I) Area (H) and diameter (I) of apoptotic tumor vessels vs all tumor vessels from segmented thick 80 μm MC38 tumor sections (n=4 biological replicates).


( J - K ) Representative immunofluorescence image of control ( J ) and 15 Gy IR treated ( K ) MC38 and B16F10 tumors stained for proliferation ( EdU ) , EC nuclei ( anti - ERG ) and tumor vessels ( anti - CD31 ) . ( L - M ) Quantification of proliferating cells ( L ) and proliferating TECs ( M ) in MC38 tumors ( n = 8 biological replicates per group ) . ( N - O ) Quantification of proliferating cells ( N ) and proliferating TECs ( O ) in B16F10 tumors ( n = 4 to 9 biological replicates per group ) . ( P - Q ) Quantification of proliferating cells ( P ) and TECs ( Q ) in MC38 tumors after fractionated IR ( n = 5 - 7 biological replicates per group ) . ( R - S ) Quantification of proliferating cells ( R ) and TECs ( S ) in B16F10 tumors after fractionated IR ( n = 5 - 7 biological replicates per group ) . ( T - U ) Measurement of proliferating TECs in ( T ) MC38 tumors ( n = 9 biological replicates ) and ( U ) B16F10 tumors ( n = 5 biological replicates ) in small ( area < 250 µm2 ) and large ( area < 250 µm2 ) tumor blood vessels .

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

(J-K) Representative immunofluorescence image of control (J) and 15 Gy IR treated (K) MC38 and B16F10 tumors stained for proliferation (EdU), EC nuclei (anti-ERG) and tumor vessels (anti-CD31). (L-M) Quantification of proliferating cells (L) and proliferating TECs (M) in MC38 tumors (n= 8 biological replicates per group). (N-O) Quantification of proliferating cells (N) and proliferating TECs (O) in B16F10 tumors (n= 4 to 9 biological replicates per group). (P-Q) Quantification of proliferating cells (P) and TECs (Q) in MC38 tumors after fractionated IR (n=5-7 biological replicates per group). (R-S) Quantification of proliferating cells (R) and TECs (S) in B16F10 tumors after fractionated IR (n= 5-7 biological replicates per group). (T-U) Measurement of proliferating TECs in (T) MC38 tumors (n= 9 biological replicates) and (U) B16F10 tumors (n= 5 biological replicates) in small (area <250 µm2) and large (area <250 µm2) tumor blood vessels.


Fluorescence was observed in the transformed N . benthamiana leaf epidermal cells , which results from complementation of the N - terminal part of the YFP fused with ATG8a ( ATG8a - N - YFP ) by the C - terminal part of the YFP fused with NBR1 ( NBR1 - C - YFP ) . No fluorescence was observed when ATG8a - N - YFP was coexpressed with unfused C - YFP or with mNBR1 - C - YFP or when unfused N - YFP was coexpressed with NBR1 - C - YFP . YFP epifluorescence images , bright - field images and overlay images of the same cells are 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 B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence was observed in the transformed N. benthamiana leaf epidermal cells, which results from complementation of the N-terminal part of the YFP fused with ATG8a (ATG8a-N-YFP) by the C-terminal part of the YFP fused with NBR1 (NBR1-C-YFP). No fluorescence was observed when ATG8a-N-YFP was coexpressed with unfused C-YFP or with mNBR1-C-YFP or when unfused N-YFP was coexpressed with NBR1-C-YFP. YFP epifluorescence images, bright-field images and overlay images of the same cells are shown.


Five weeks - old Arabidopsis wild - type Col - 0 plants were placed in the 22 ° C and 45 ° C growth chambers and total RNA was isolated from leaf samples collected at indicated times . Transcript levels were determined using real - time qRT - PCR . Error bars indicate SE ( n = 3 ) .

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

Five weeks-old Arabidopsis wild-type Col-0 plants were placed in the 22°C and 45°C growth chambers and total RNA was isolated from leaf samples collected at indicated times. Transcript levels were determined using real-time qRT-PCR. Error bars indicate SE (n = 3).


( A ) Four - weeks old transgenic wild - type Col - 0 ( WT ) and atg7 - 2 mutant plants expressing GFP - ATG8a were treated with ( 45 ° C ) or without ( 22 ° C ) heat shock for 3 h and then placed at room temperature for 0 . 5 h . The leaves were visualized by fluorescence confocal microscopy of GFP signal . ( B ) Numbers of punctate GFP - ATG8a spots representing autophagosomes per 10 , 000 µm2 section . Means and SE were calculated from three experiments . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means do not differ significantly if they are indicated with the same letter . Bar = 20 µm .

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

(A) Four-weeks old transgenic wild-type Col-0 (WT) and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.


( Top panel ) Four - weeks old transgenic wild - type Col - 0 ( WT ) , atg7 - 2 and nbr1 - 1 mutant plants expressing GFP - NBR1 were treated with ( 45 ° C ) or without ( 22 ° C ) heat shock for 3 h and then placed at room temperature for 0 . 5 h . The leaves were visualized by fluorescence confocal microscopy of GFP signal . ( Bottom panel ) Numbers of punctate GFP - NBR1 spots per 10 , 000 µm2 section . Means and SE were calculated from three experiments . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means do not differ significantly if they are indicated with the same letter . Bar = 20 µm .

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

(Top panel) Four-weeks old transgenic wild-type Col-0 (WT), atg7-2 and nbr1-1 mutant plants expressing GFP-NBR1 were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (Bottom panel) Numbers of punctate GFP-NBR1 spots per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.


( A ) Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 mutant plants were placed in 22 ° C and 45 ° C growth for 10 hours and then moved to room temperature for 3 - day recovery or ( B ) sprayed with 20 µM methyl viologen ( MV ) and kept under light for two days before the picture of representative plants was taken . The experiments were repeated three times with similar results .

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

(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth for 10 hours and then moved to room temperature for 3-day recovery or (B) sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.


( B ) Fv / Fm images . ( C ) Average values for the Fv / Fm images . Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 mutant plants were placed in 22 ° C and 45 ° C growth chambers . Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv / Fm . Error bars in ( A ) and ( C ) indicate SE ( n = 5 ) . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means of EL or Fv / Fm do not differ significantly if they are indicated with the same letter . The experiments were repeated twice with similar results .

O O O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fv/Fm images. (C) Average values for the Fv/Fm images. Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth chambers. Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv/Fm. Error bars in (A) and (C) indicate SE (n = 5). According to Duncan's multiple range test (P = 0.05), means of EL or Fv/Fm do not differ significantly if they are indicated with the same letter. The experiments were repeated twice with similar results.


( A ) Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50 % humidity . The photograph of representative plants was taken 10 days after withholding watering . The experiment was repeated twice with similar results .

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

(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50% humidity. The photograph of representative plants was taken 10 days after withholding watering. The experiment was repeated twice with similar results.


( B ) Seven days - old seedlings Col - 0 WT , atg5 , atg7 and nbr1 grown on solid MS medium were transferred to the same medium ( control ) or the same medium containing 0 . 16 M NaCl and photographed 5 days later . The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments . According to Duncan ' s multiple range test ( P = 0 . 05 ) , means do not differ significantly if they are indicated with the same letter .

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

(B) Seven days-old seedlings Col-0 WT, atg5, atg7 and nbr1 grown on solid MS medium were transferred to the same medium (control) or the same medium containing 0.16 M NaCl and photographed 5 days later. The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter.


( A ) Eight weeks old wild type ( WT ) , atg5 , atg7 and nbr1 plants grown under normal growth conditions in a growth chamber . ( B ) Four weeks old WT , atg5 , atg7 and nbr1 plants after being kept in the dark for 6 days . ( C ) Percentage of plants that survived the various lengths in dark as determined by resumption of growth . Each point represents the average of 10 plants . The experiments were repeated twice with similar results .

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

(A) Eight weeks old wild type (WT), atg5, atg7 and nbr1 plants grown under normal growth conditions in a growth chamber. (B) Four weeks old WT, atg5, atg7 and nbr1 plants after being kept in the dark for 6 days. (C) Percentage of plants that survived the various lengths in dark as determined by resumption of growth. Each point represents the average of 10 plants. The experiments were repeated twice with similar results.


( A ) Responses of Col - 0 wild type ( WT ) , atg5 , atg7 and nbr1 plants to Botrytis . Wild - type and mutant plants were inoculated by spraying with spore suspension at a density of 2 . 5 × 105 spores ml − 1 , and kept at high humidity . Pictures of representative plants were taken at 4 and 5 days post inoculation ( dpi ) . ( B ) Quantitative real - time PCR analysis of the B . cinerea ActA ( BcActA ) transcript levels in infected Arabidopsis plants at 5 dpi . The experiments were repeated twice with similar results .

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

(A) Responses of Col-0 wild type (WT), atg5, atg7 and nbr1 plants to Botrytis. Wild-type and mutant plants were inoculated by spraying with spore suspension at a density of 2.5×105 spores ml−1, and kept at high humidity. Pictures of representative plants were taken at 4 and 5 days post inoculation (dpi). (B) Quantitative real-time PCR analysis of the B. cinerea ActA (BcActA) transcript levels in infected Arabidopsis plants at 5 dpi. The experiments were repeated twice with similar results.


( A ) Diagrams of wild - type and mutant NBR1 proteins . The conserved W and I residues in the LIR motif between the two UBA domains were changed to A residues in the NBR1W661A / I664A ( mNBR1 ) mutant protein . ( B ) Five weeks - old Arabidopsis Col - 0 wild type ( WT ) , nbr1 and transgenic nbr1 plants expressing the wild - type NBR1 ( lines 3 and 6 ; see Figure S2 ) or the NBR1W661A / I664A ( mNBR1 ) ( lines 2 and 6 ; see Figure S2 ) mutant gene were placed in a 45 ° C growth chambers for 10 hours and then moved to room temperature for 3 - day recovery before the picture as taken . The experiment was repeated three times with similar results .

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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O

(A) Diagrams of wild-type and mutant NBR1 proteins. The conserved W and I residues in the LIR motif between the two UBA domains were changed to A residues in the NBR1W661A/I664A (mNBR1) mutant protein. (B) Five weeks-old Arabidopsis Col-0 wild type (WT), nbr1 and transgenic nbr1 plants expressing the wild-type NBR1 (lines 3 and 6; see Figure S2) or the NBR1W661A/I664A (mNBR1) (lines 2 and 6; see Figure S2) mutant gene were placed in a 45°C growth chambers for 10 hours and then moved to room temperature for 3-day recovery before the picture as taken. The experiment was repeated three times with similar results.


Leaf tissues from wild type ( WT ) and atg7 expressing NBR1 - TAP were collected at indicated hours ( h ) under 45 ° C and prepared for soluble and insoluble proteins as described in Materials and Methods . Proteins from the first supernatants ( S ) and last pellets ( P ) were subjected to SDS - PAGES and probed with a peroxidase - conjugated anti - peroxidase antibody for detection of NBR1 - TAP . Protein samples prepared from non - transgenic Col - 0 wild - type plants were included as controls .

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

Leaf tissues from wild type (WT) and atg7 expressing NBR1-TAP were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with a peroxidase-conjugated anti-peroxidase antibody for detection of NBR1-TAP. Protein samples prepared from non-transgenic Col-0 wild-type plants were included as controls.


( A ) Accumulation of insoluble proteins . Leaf tissues from wild - type ( WT ) , atg7 and nbr1 mutants collected at indicated hours ( h ) under 45 ° C for preparation of total , soluble and insoluble proteins as described in Materials and Methods . Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated . ( B ) Profiles of soluble and insoluble proteins . Proteins from the first supernatants ( S ) and last pellets ( P ) were subjected to SDS - PAGES and stained with Coomassie brilliant blue . Major proteins accumulated in the pellets from heat - stressed atg7 mutant plants were indicated by arrows .

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

(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT), atg7 and nbr1 mutants collected at indicated hours (h) under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated. (B) Profiles of soluble and insoluble proteins. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and stained with Coomassie brilliant blue. Major proteins accumulated in the pellets from heat-stressed atg7 mutant plants were indicated by arrows.


Leaf tissues from wild type ( WT ) , atg7 and nbr1 mutants were collected at indicated hours ( h ) under 45 ° C and prepared for soluble and insoluble proteins as described in Materials and Methods . Proteins from the first supernatants ( S ) and last pellets ( P ) were subjected to SDS - PAGES and probed with anti - ubiquitin monoclonal antibody . The experiment was repeated three times with similar results .

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

Leaf tissues from wild type (WT), atg7 and nbr1 mutants were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.


B . Immunoblot shows GST - GGA - GAT domain bound Arf1 ( active ; top ) and total Arf1 ( input lysates ; bottom ) from equal aliquots of lysates of HeLa cells that were stimulated with EGF for the indicated time points prior to lysis .

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

B. Immunoblot shows GST-GGA-GAT domain bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of HeLa cells that were stimulated with EGF for the indicated time points prior to lysis.


C . Graphs display the model fitting for Arf1 activation dynamics . The experimentally determined Arf1 activation ( in B ) dynamics are displayed as black dots with error bars , representing mean ± S . E . M ( n = 3 biological replicates ) , and numerical simulation is shown by the blue continuous line .

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

C. Graphs display the model fitting for Arf1 activation dynamics. The experimentally determined Arf1 activation (in B) dynamics are displayed as black dots with error bars, representing mean ± S.E.M (n=3 biological replicates), and numerical simulation is shown by the blue continuous line.


E . HeLa cells expressing Arf1 - HA were serum starved overnight ( E , top ) and subsequently stimulated with EGF for 5 min ( E , bottom ) prior to fixation with PFA . Fixed cells were stained for Arf1 ( HA ; green ) and GIV ( red ) and nuclei ( DAPI ; blue ) . Panels on the left show overlay of all 3 stains and representative RGB plots of sections through the Arf1 - stained pixels . Panels on the right display the magnified 3D surface plots of the boxed regions in the left panels . Scale bar = 10 µm .

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

E. HeLa cells expressing Arf1-HA were serum starved overnight (E, top) and subsequently stimulated with EGF for 5 min (E, bottom) prior to fixation with PFA. Fixed cells were stained for Arf1 (HA; green) and GIV (red) and nuclei (DAPI; blue). Panels on the left show overlay of all 3 stains and representative RGB plots of sections through the Arf1-stained pixels. Panels on the right display the magnified 3D surface plots of the boxed regions in the left panels. Scale bar = 10 µm.


C . Equal aliquots ( ~ 45 µg ) of whole cell lysates of control ( shControl ; top ) and GIV - GEM depleted ( shGIV ; bottom ) HeLa cells were analyzed for GIV and tubulin ( loading control ) by immunoblotting ( IB ) .

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C. Equal aliquots (~45 µg) of whole cell lysates of control (shControl; top) and GIV-GEM depleted (shGIV; bottom) HeLa cells were analyzed for GIV and tubulin (loading control) by immunoblotting (IB).


D . Control ( sh Control ; top ) and GIV - GEM depleted ( shGIV ; bottom ) HeLa cells were co - transfected with Gαi1 - YFP , Gβ1 - CFP and Gγ2 ( untagged ) and live cells were analyzed by FRET imaging at steady - state , after being serum starved in 0 . 2 % FBS overnight and then after stimulation with 50 nM EGF . Representative freeze - frame FRET images are shown . FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel . FRET scale is shown in the inset . Golgi and PM regions of interest are indicated with arrows . Scale bar = 10 µm . See also Figure EV2A - B for free - frame images for additional time points in control HeLa cells .

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 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 O O O O O

D. Control (sh Control; top) and GIV-GEM depleted (shGIV; bottom) HeLa cells were co-transfected with Gαi1-YFP, Gβ1-CFP and Gγ2 (untagged) and live cells were analyzed by FRET imaging at steady-state, after being serum starved in 0.2% FBS overnight and then after stimulation with 50 nM EGF. Representative freeze-frame FRET images are shown. FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel. FRET scale is shown in the inset. Golgi and PM regions of interest are indicated with arrows. Scale bar = 10 µm. See also Figure EV2A-B for free-frame images for additional time points in control HeLa cells.


F . HeLa cells starved with 0 . 2 % FBS overnight or stimulated subsequently with 50 nM EGF were fixed and stained for active Gαi ( green ; anti - Gαi : GTP mAb ) and Man II ( red ) and analyzed by confocal microscopy . Activation of Gαi was detected exclusively after EGF stimulation . When detected , active Gαi colocalizes with Man II ( yellow pixels in merge panel ) . See also Figure EV2C - D for additional time points and stimulus . Scale bar = 7 . 5 µm .

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F. HeLa cells starved with 0.2% FBS overnight or stimulated subsequently with 50 nM EGF were fixed and stained for active Gαi (green; anti-Gαi:GTP mAb) and Man II (red) and analyzed by confocal microscopy. Activation of Gαi was detected exclusively after EGF stimulation. When detected, active Gαi colocalizes with Man II (yellow pixels in merge panel). See also Figure EV2C-D for additional time points and stimulus. Scale bar = 7.5 µm.


G . Model fit for the fold change of active tGTPase ( denoted as tG * ) . Experiment data is the fold change of ∆ FRET / CFP in D and is shown as mean ± S . E . M ( n = 8 ; 3 - 5 cells from 2 independent biological experiments ) . Continuous lines display the model simulation results after parameter

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Model fit for the fold change of active tGTPase (denoted as tG*). Experiment data is the fold change of ∆FRET/CFP in D and is shown as mean ± S.E.M (n=8; 3-5 cells from 2 independent biological experiments). Continuous lines display the model simulation results after parameter fitting


I . Immunoblot shows bound Arf1 ( active ; top ) and total Arf1 ( input lysates ; bottom ) from equal aliquots of lysates of control ( sh Control ) and GIV - depleted ( shGIV ) HeLa cells . Cells were stimulated with EGF for the indicated time points prior to lysis . Bar graphs in Figure EV2E display the fold change in Arf1 activity normalized to t0 min . ' Low ' and ' high ' indicate exposures .

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

I. Immunoblot shows bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of control (sh Control) and GIV-depleted (shGIV) HeLa cells. Cells were stimulated with EGF for the indicated time points prior to lysis. Bar graphs in Figure EV2E display the fold change in Arf1 activity normalized to t0 min. 'Low' and 'high' indicate exposures.


D - E . The secretion ( D ) or the cell number ( E ) as a function of stimulus in coupled and uncoupled switches . The stimulus = 0 , 0 . 00046 , 0 . 0046 , 0 . 046 , 0 . 115 , 0 . 23 and 0 . 46 correspond to varying doses of EGF in simulations , ranging from 0 , 0 . 1 nM , 1 nM , 10 nM , 25 nM , 50 nM , and 100 nM , respectively . The error bar denotes the S . D based on 1000 repeated independent simulations of ODEs when noise is in the stimulus and connections .

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

D-E. The secretion (D) or the cell number (E) as a function of stimulus in coupled and uncoupled switches. The stimulus =0, 0.00046, 0.0046, 0.046, 0.115, 0.23 and 0.46 correspond to varying doses of EGF in simulations, ranging from 0, 0.1 nM, 1 nM, 10 nM, 25 nM, 50 nM, and 100 nM, respectively. The error bar denotes the S.D based on 1000 repeated independent simulations of ODEs when noise is in the stimulus and connections.


F . The bar plot depicts cell numbers achieved by cells with either coupled or uncoupled switches , at different levels of stimulus . For the first two bars , the height and error bars are the mean and S . D . of cell number when stimulus = 0 . 046 in E , respectively . For the last two bars , the height and error bars are the mean and S . D . of cell number when stimulus = 0 in E , 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 O O O O O O O

F. The bar plot depicts cell numbers achieved by cells with either coupled or uncoupled switches, at different levels of stimulus. For the first two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0.046 in E, respectively. For the last two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0 in E, respectively.


B . Immunoblots showing intracellular ( left ) and secreted ( in the media ; right ) GFP - MMP9 at 24 h after stimulation with varying doses of EGF . Tubulin , used as a loading control , confirms the presence of a similar number of plated cells in the assay .

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B. Immunoblots showing intracellular (left) and secreted (in the media; right) GFP-MMP9 at 24 h after stimulation with varying doses of EGF. Tubulin, used as a loading control, confirms the presence of a similar number of plated cells in the assay.


C . Left : Graph displays experimentally determined secretion of GFP - MMP9 in response to varying doses of EGF in control ( shControl ) and GIV - depleted ( shGIV ) HeLa cells ( as in B ) , and quantified by band densitometry . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . p values were determined by two - sided unpaired t - test . Right : Schematic diagram of dose responses ( mG * and secretion ) for the single switch and coupled switches . Coupled switches stretch the range of proportionate responses . Single mG switch results in misaligned responses . DoRA , dose response alignment .

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C. Left: Graph displays experimentally determined secretion of GFP-MMP9 in response to varying doses of EGF in control (shControl) and GIV-depleted (shGIV) HeLa cells (as in B), and quantified by band densitometry. Results are expressed as mean ± S.E.M; n = 3 biological replicates. p values were determined by two-sided unpaired t-test. Right: Schematic diagram of dose responses (mG* and secretion) for the single switch and coupled switches. Coupled switches stretch the range of proportionate responses. Single mG switch results in misaligned responses. DoRA, dose response alignment.


G - H . Control ( parental ) and GIV - depleted ( GIV KO ) HeLa cells grown in different concentrations of serum ( FBS % ) were treated or not with varying concentrations of BFA ( µM ) as indicated . Line graphs in 3D ( G ) depict the formazan absorbance expressed as a measure of cell viability from the HeLa cells in various conditions tested . Bar graphs ( H ) depict the cell number in serum - free growth conditions that are supported exclusively by autocrine secrete - and - sense loop ( without BFA ; BFA = 0 . 0 µM ) or when such loop is interrupted ( BFA = 0 . 1 µM ) . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . Statistical significance was determined by one way ANOVA .

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G-H. Control (parental) and GIV-depleted (GIV KO) HeLa cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as indicated. Line graphs in 3D (G) depict the formazan absorbance expressed as a measure of cell viability from the HeLa cells in various conditions tested. Bar graphs (H) depict the cell number in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA.


I - K . Control ( parental ) and GIV - depleted ( GIV KO ) MDA MB - 231 cells grown in different concentrations of serum ( FBS % ) were treated or not with varying concentrations of BFA ( µM ) as in G - H . Line graphs in 3D ( I ) depict the formazan absorbance expressed as a measure of cell viability from the MDA MB - 231 cells in various conditions tested . Bar graphs ( J ) depict the viability of the MDA MB - 231 cells in serum - free growth conditions that are supported exclusively by autocrine secrete - and - sense loop ( without BFA ; BFA = 0 . 0 µM ) or when such loop is interrupted ( BFA = 0 . 1 µM ) . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . Statistical significance was determined by one way ANOVA . Immunoblots ( K ) of equal aliquots of whole cell lysates confirm the depletion of GIV compared to tubulin ( loading control ) . See also Appendix Figure S5D - H for dot plots and early and late apoptotic fractions . Results are expressed as mean ± S . E . M ; n = 3 biological replicates .

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I-K. Control (parental) and GIV-depleted (GIV KO) MDA MB-231 cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as in G-H. Line graphs in 3D (I) depict the formazan absorbance expressed as a measure of cell viability from the MDA MB-231 cells in various conditions tested. Bar graphs (J) depict the viability of the MDA MB-231 cells in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA. Immunoblots (K) of equal aliquots of whole cell lysates confirm the depletion of GIV compared to tubulin (loading control). See also Appendix Figure S5D-H for dot plots and early and late apoptotic fractions. Results are expressed as mean ± S.E.M; n = 3 biological replicates.


Quantification of mitotic defects as in ( B ) of H2B - mNeon - expressing HeLaEGFP - AID - CENATAC cells treated as indicated ( three or five biological replicates , > 85 cells in total per condition ) . For 2xZF the four zinc - finger cysteines were mutated to alanines ; for Δ1 - 4 the corresponding motif was removed .

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Quantification of mitotic defects as in (B) of H2B-mNeon-expressing HeLaEGFP-AID-CENATAC cells treated as indicated (three or five biological replicates, >85 cells in total per condition). For 2xZF the four zinc-finger cysteines were mutated to alanines; for Δ1-4 the corresponding motif was removed.


Representative stills of HeLaEGFP - AID - CENATAC cells expressing H2B - mNeon and depleted of CENATAC . Microtubules were visualized with SiR - Tubulin . Arrowheads and arrows indicate non - congressed chromosomes and supernumerary spindle poles , respectively . Scale bar , 5 μm . Time in hours . See Figure EV2 for the control condition . See also Movies EV1 and EV2 . IAA , 3 - indoleacetic acid .

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Representative stills of HeLaEGFP-AID-CENATAC cells expressing H2B-mNeon and depleted of CENATAC. Microtubules were visualized with SiR-Tubulin. Arrowheads and arrows indicate non-congressed chromosomes and supernumerary spindle poles, respectively. Scale bar, 5 μm. Time in hours. See Figure EV2 for the control condition. See also Movies EV1 and EV2. IAA, 3-indoleacetic acid.


Graph of fold - changes of proteins enriched ( P - value < 0 . 05 ) in proteomics analysis of CENATAC vs . control co - immunoprecipitations of HeLaEGFP - CENATAC cells ( three biological replicates ) . Splicing factors are depicted in orange . See also Appendix Figure S7 .

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Graph of fold-changes of proteins enriched (P-value < 0.05) in proteomics analysis of CENATAC vs. control co-immunoprecipitations of HeLaEGFP-CENATAC cells (three biological replicates). Splicing factors are depicted in orange. See also Appendix Figure S7.


Glycerol gradient ( 10 - 30 % ) analysis of HeLa S3 nuclear extracts . snRNAs were detected by Northern blot analysis , proteins ( CENATAC and PRPF4 ) by western blot . Locations of the U6atac mono - snRNP , U4atac / U6atac di - snRNP and U4atac / U6atac . U5 tri - snRNP are indicated .

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Glycerol gradient (10-30%) analysis of HeLa S3 nuclear extracts. snRNAs were detected by Northern blot analysis, proteins (CENATAC and PRPF4) by western blot. Locations of the U6atac mono-snRNP, U4atac/U6atac di-snRNP and U4atac/U6atac.U5 tri-snRNP are indicated.


RT - PCRs of IPO5 , SUDS3 and ZCCHC8 minor and adjacent major introns on RNA extracted from patient lymphoblasts or HeLaEGFP - AID - CENATAC cells depleted of GAPDH or CENATAC as indicated . u , unspliced ; s , spliced .

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RT-PCRs of IPO5, SUDS3 and ZCCHC8 minor and adjacent major introns on RNA extracted from patient lymphoblasts or HeLaEGFP-AID-CENATAC cells depleted of GAPDH or CENATAC as indicated. u, unspliced; s, spliced.


RT - PCRs of ZRSR2 and GAPDH ( to visualize ZRSR2 knockdown efficiency , upper ) and RT - PCRs of SUDS3 minor intron 7 ( middle ) and major intron 5 ( lower ) on RNA extracted from HeLaEGFP - AID - CENATAC or DLD - 1 cells depleted of GAPDH or ZRSR2 as indicated .

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RT-PCRs of ZRSR2 and GAPDH (to visualize ZRSR2 knockdown efficiency, upper) and RT-PCRs of SUDS3 minor intron 7 (middle) and major intron 5 (lower) on RNA extracted from HeLaEGFP-AID-CENATAC or DLD-1 cells depleted of GAPDH or ZRSR2 as indicated.


RT - PCR of major ( U2 - type ) AT - AC introns in SYCP2 ( intron 5 ) and TAF2 ( intron 1 ) , and minor ( U12 - type ) AT - AC intron in IPO5 ( intron 21 ) on RNA extracted from HeLaEGFP - AID - CENATAC cells depleted of GAPDH or CENATAC . Schematic representations of unspliced / spliced PCR products are depicted on the right .

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RT-PCR of major (U2-type) AT-AC introns in SYCP2 (intron 5) and TAF2 (intron 1), and minor (U12-type) AT-AC intron in IPO5 (intron 21) on RNA extracted from HeLaEGFP-AID-CENATAC cells depleted of GAPDH or CENATAC. Schematic representations of unspliced/spliced PCR products are depicted on the right.


RT - PCR P120 reporter assay ( Hall & Padgett , 1996 ) to measure the relative usage of A - type ( AT - AC ) and G - type ( GT - AG ) 5 ' splice sites in direct competition . Upper : schematic diagram showing the overall architecture of the reporter construct with its down - and upstream splice site ( thick red and blue bars , respectively ) and the products created by splicing ( Dn and Up , respectively ) . Lower : RT - PCRs of the reporter with A - or G - type splice sites in the down - or upstream positions as indicated below the gel . SS , splice site . * , PCR product after use of a cryptic major splice site ( not shown in the schematic ) .

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RT-PCR P120 reporter assay(Hall & Padgett, 1996) to measure the relative usage of A-type (AT-AC) and G-type (GT-AG) 5' splice sites in direct competition. Upper: schematic diagram showing the overall architecture of the reporter construct with its down- and upstream splice site (thick red and blue bars, respectively) and the products created by splicing (Dn and Up, respectively). Lower: RT-PCRs of the reporter with A- or G-type splice sites in the down- or upstream positions as indicated below the gel. SS, splice site. *, PCR product after use of a cryptic major splice site (not shown in the schematic).


A . Intracellular survival of GAS strains 188 , 188SLO - ( SLO - ) , and 188NADase - ( NADase - ) . Intracellular CFU were quantified at 2 h , 4 h , 6 h , 8 h , and 24 h , and intracellular survival was calculated as the percent viable CFU compared to 2 h .

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A. Intracellular survival of GAS strains 188, 188SLO- (SLO-), and 188NADase- (NADase-). Intracellular CFU were quantified at 2 h, 4 h, 6 h, 8 h, and 24 h, and intracellular survival was calculated as the percent viable CFU compared to 2 h.


B . Intracellular survival of GAS strain 188SLO - that was partially complemented from plasmid piSLO ( SLO - ( piSLO ) ) , strain 188 and 188SLO - that contained the empty complementation vector , 188 ( pSIV4 ) and SLO - ( pSIV4 ) , respectively .

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B. Intracellular survival of GAS strain 188SLO- that was partially complemented from plasmid piSLO (SLO-(piSLO)), strain 188 and 188SLO- that contained the empty complementation vector, 188(pSIV4) and SLO-(pSIV4), respectively.


C . Intracellular survival of GAS strain JRS4 , JRS4SLO - ( SLO - ) , and JRS4NADase - ( NADase - ) . Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD . * , P < 0 . 001 .

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C. Intracellular survival of GAS strain JRS4, JRS4SLO- (SLO-), and JRS4NADase- (NADase-). Experiments were performed in triplicate and values represent the mean of three independent experiments ± SD. *, P<0.001.


A . Confocal microscopy of the association between EGFP - LC3 and GAS strain 188 , 188SLO - ( SLO - ) , 188SLS - ( SLS - ) , or 188SLO - SLS - ( SLO - SLS - ) . Immunofluorescent staining distinguished intracellular ( Alexa - 568 , red ) from extracellular ( Alexa - 568 and Alexa - 660 , red and blue , respectively ) GAS . Scale bar = 10 µm . The percent of intracellular GAS that were associated with EGFP - LC3 at 3 h post - infection is shown for each strain .

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A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain.


B . Electron microscopy of GAS strains 188 , 188SLO - , and 188SLO - SLS - at 3 h post - infection . Arrowheads indicate the presence of double or multiple membranes ( 188 and 188SLO - ) or single membranes ( 188SLO - SLS - ) partially or completely surrounding bacteria . Vacuolar compartments associated with 188 and 188SLO - also contain cytosolic material .

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B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material.


C . Intracellular survival of GAS strains 188 , 188SLO - , 188SLS - , and 188SLO - SLS - . * , P < 0 . 05 .

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C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05.


D . Intracellular survival of 188 or 188SLO - in the presence or absence of Beclin1 knockdown .

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D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.


A . Intracellular survival of GAS strains 188 , 188NADase - , and 188NADase ( G330D ) . Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20 - to 40 - fold reduction in intracellular survival compared to parent strain 188 . * , P < 0 . 001 .

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A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001.


and activity measurements ( C ) of culture supernatants from GAS strains 188 , 188NADase - , and 188NADase ( G330D ) .

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and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).


A . Confocal microscopy demonstrating the association between ubiquitin ( green ) and intracellular GAS ( red ) at 30 min post - infection in keratinocytes infected with GAS strain 188 , 188SLO - , 188NADase - , or 188SLO ( Y255A ) . Extracellular GAS were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments .

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A. Confocal microscopy demonstrating the association between ubiquitin (green) and intracellular GAS (red) at 30 min post-infection in keratinocytes infected with GAS strain 188, 188SLO-, 188NADase-, or 188SLO(Y255A). Extracellular GAS were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments.


B . Hemolytic activity of culture supernatants of GAS strain 188 , 188SLO - , and 188SLO ( Y255A ) . The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane , demonstrated by the failure of culture supernatants from 188SLO ( Y255A ) to lyse erythrocytes despite producing wild - type amounts of the variant SLO protein . Inset , corresponding Western blot for SLO in culture supernatants from these strains .

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B. Hemolytic activity of culture supernatants of GAS strain 188, 188SLO-, and 188SLO(Y255A). The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane, demonstrated by the failure of culture supernatants from 188SLO(Y255A) to lyse erythrocytes despite producing wild-type amounts of the variant SLO protein. Inset, corresponding Western blot for SLO in culture supernatants from these strains.


. C . NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771 , 771SLO - , 771SLO ( Y255A ) , or 771NADase - . Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO ( Y255A ) , but reduced in 771SLO - .

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. C. NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771, 771SLO-, 771SLO(Y255A), or 771NADase-. Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO(Y255A), but reduced in 771SLO-.


D . The intracellular survival of 188SLO ( Y255A ) was significantly lower than that of its parental strain , 188 , and similar to that of 188SLO - . * , P < 0 . 01 .

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D. The intracellular survival of 188SLO(Y255A) was significantly lower than that of its parental strain, 188, and similar to that of 188SLO-. *, P<0.01.


E . Confocal microscopy demonstrating the association between galectin 8 ( green ) and intracellular GAS ( red ) in keratinocytes infected for 30 min with GAS strain 188 , 188SLO - , or 188 SLO - SLS - . Extracellular GAS ( GAS ( out ) ) were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments . 188SLO - SLS - was never associated with galectin 8 ( 0 % ) .

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E. Confocal microscopy demonstrating the association between galectin 8 (green) and intracellular GAS (red) in keratinocytes infected for 30 min with GAS strain 188, 188SLO-, or 188 SLO-SLS-. Extracellular GAS (GAS(out)) were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments. 188SLO-SLS- was never associated with galectin 8 (0%).


SLO and NADase inhibit lysosomal fusion to GAS - containing autophagosomes in oropharyngeal keratinocytes . A . Confocal microscopy of keratinocytes infected with GAS strain 188 , 188SLO - , or 188NADase - demonstrating the association of the lysosomal marker LAMP - 1 ( red ) with GAS ( blue ) contained within EGFP - LC3 ( green ) - positive compartments at 1 h , 3 h , and 6 h post - infection . Scale bar = 10 µm . B . Quantification of the percent of GAS within EGFP - LC3 - positive compartments that are co - localized with LAMP - 1 at 1 h , 3 h , and 6 h . Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment .

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SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.


( A , B ) Fluorescence confocal microscopy images show TMRE staining and GFP : : MIRO - 1 in WT and fzo - 1 mutants animals . Dashed lines outline regions where fluorescence intensity was quantified .

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(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT and fzo-1 mutants animals. Dashed lines outline regions where fluorescence intensity was quantified.


( C ) Immunoblot shows protein levels of MIRO - 1 in WT and fzo - 1 mutant animals , with tubulin and COX - 4 serving as loading controls .

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(C) Immunoblot shows protein levels of MIRO-1 in WT and fzo-1 mutant animals, with tubulin and COX-4 serving as loading controls.


( D ) Fluorescence confocal images show GFP : : MIRO - 1 and mito : : mKate2 in WT and fzo - 1 mutants . ( E ) Quantification of MIRO - 1 : : GFP levels in WT and fzo - 1 mutants . n ≥ 33 , biological replicates .

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(D) Fluorescence confocal images show GFP::MIRO-1 and mito::mKate2 in WT and fzo-1 mutants. (E) Quantification of MIRO-1::GFP levels in WT and fzo-1 mutants. n≥33, biological replicates.


( A , B ) Fluorescence confocal microscopy images show TMRE staining and GFP : : MIRO - 1 in WT animals with and without needle wounding . Dashed lines outline regions where fluorescence intensity was quantified .

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(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT animals with and without needle wounding. Dashed lines outline regions where fluorescence intensity was quantified.


( C ) Representative confocal images showg GFP : : MIRO - 1 and mito : : mKate2 before and 2 hours after wounding .

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(C) Representative confocal images showg GFP::MIRO-1 and mito::mKate2 before and 2 hours after wounding.


( D ) Immunoblot shows protein levels of MIRO - 1 before and 2 hours after wounding , with tubulin and COX - 4 serving as loading controls .

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(D) Immunoblot shows protein levels of MIRO-1 before and 2 hours after wounding, with tubulin and COX-4 serving as loading controls.


( A ) Representative confocal images show the TMRE and mito : : GFP signals in WT , miro - 1 ( tm1966 ) , miro - 1 ( ΔEF ) ( EF - hand domain mutation ) , and miro - 1 ( OE , overexpression ) animals . ( B ) Quantitation of TMRE and mito : : GFP signal ( normalized ratio of TMRE fluorescence to mito : : GFP fluorescence ) . n > 200 mitochondria .

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(A) Representative confocal images show the TMRE and mito::GFP signals in WT, miro-1(tm1966), miro-1(ΔEF) (EF-hand domain mutation), and miro-1(OE, overexpression) animals. (B) Quantitation of TMRE and mito::GFP signal (normalized ratio of TMRE fluorescence to mito::GFP fluorescence). n > 200 mitochondria.


( D ) ATP content of crude isolated mitochondria from WT and miro - 1 ( tm1966 ) mutants as measured with a commercial luminescence kit . n = 3 , biological replicates .

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(D) ATP content of crude isolated mitochondria from WT and miro-1(tm1966) mutants as measured with a commercial luminescence kit. n = 3, biological replicates.


( D ) Western blot analysis of the interaction between MIRO - 1 and VDAC - 1 by coimmunoprecipitating with GFP nanobody - conjugated beads in GFP : : MIRO - 1 animals and detected by GFP and VDAC - 1 antibodies .

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(D) Western blot analysis of the interaction between MIRO-1 and VDAC-1 by coimmunoprecipitating with GFP nanobody-conjugated beads in GFP::MIRO-1 animals and detected by GFP and VDAC-1 antibodies.


( E ) Western blot analysis of the interaction between MIRO - 1 and VDAC - 1 by coimmunoprecipitating with VDAC - 1 antibody - conjugated beads in GFP : : MIRO - 1 animals and detected by GFP and VDAC - 1 antibodies .

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(E) Western blot analysis of the interaction between MIRO-1 and VDAC-1 by coimmunoprecipitating with VDAC-1 antibody-conjugated beads in GFP::MIRO-1 animals and detected by GFP and VDAC-1 antibodies.


( F and G ) Immunoblot showing the immunoprecipitation of purified GST : : MIRO - 1 and VDAC - 1 : : His with GST - beads and His - beads , respectively .

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(F and G) Immunoblot showing the immunoprecipitation of purified GST::MIRO-1 and VDAC-1::His with GST-beads and His-beads, respectively.


( H ) BN - PAGE and immunoblot analysis of the total worm lysate of GFP : : MIRO - 1 ( KI ) by GFP and VDAC - 1 antibodies . Green shading highlights the complex that contained MIRO - 1 and VDAC - 1 proteins .

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(H) BN-PAGE and immunoblot analysis of the total worm lysate of GFP::MIRO-1 (KI) by GFP and VDAC-1 antibodies. Green shading highlights the complex that contained MIRO-1 and VDAC-1 proteins.


( A ) Schematic of the vdac - 1 transgene by CRISPR - Cas9 method . The images below show the young adult stage worms of WT and vdac - 1 ( zju247 ) mutants . ( B ) Representative confocal images showing TMRE staining and mito : : GFP in L4440 , vdac - 1 RNAi , vdac - 1 ( zju247 ) , and vdac - 1 OE animals .

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(A) Schematic of the vdac-1 transgene by CRISPR-Cas9 method. The images below show the young adult stage worms of WT and vdac-1(zju247) mutants. (B) Representative confocal images showing TMRE staining and mito::GFP in L4440, vdac-1 RNAi, vdac-1(zju247), and vdac-1 OE animals.


( E ) ATP content in crude isolated mitochondria of WT animals and vdac - 1 ( zju247 ) mutants . Quantification is conducted with a commercial luminescence kit . n = 3 , biological replicates .

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(E) ATP content in crude isolated mitochondria of WT animals and vdac-1(zju247) mutants. Quantification is conducted with a commercial luminescence kit. n = 3, biological replicates.


( B ) Immunoprecipitation of GFP : : MIRO - 1 and GFP : : MIRO - 1 ( E473G ) with GFP nanobody - conjugated beads .

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(B) Immunoprecipitation of GFP::MIRO-1 and GFP::MIRO-1(E473G) with GFP nanobody-conjugated beads.


( F ) Immunoprecipitation of GFP : : MIRO - 1 in fzo - 1 mutant with GFP nanobody - conjugated beads . The interaction between GFP : : MIRO - 1 and VDAC - 1 is increased in fzo - 1 mutants .

(F) Immunoprecipitation of GFP::MIRO-1 in fzo-1 mutant with GFP nanobody-conjugated beads. The interaction between GFP::MIRO-1 and VDAC-1 is increased in fzo-1 mutants.


( G ) BN - PAGE and immunoblot analysis of VDAC - 1 oligomerization crosslinked with BMH in WT and miro - 1 ( tm1966 ) mutants using VDAC - 1 antibody .

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(G) BN-PAGE and immunoblot analysis of VDAC-1 oligomerization crosslinked with BMH in WT and miro-1(tm1966) mutants using VDAC-1 antibody.


( A ) RFP - ATG8g - labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ ( oD600 = 0 . 2 ) at 2 dpi in the presence or absence of ConA ( bars = 20 μm ) . Puncta were calculated from z - stacks ( 15 ) of n = 6 individuals using ImageJ . Central band of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Different letters indicate statistically different groups ( P < 0 . 05 ) as determined by one - way ANOVA . The experiment was repeated twice with similar results .

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(A) RFP-ATG8g-labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ (oD600=0.2) at 2 dpi in the presence or absence of ConA (bars = 20 μm). Puncta were calculated from z-stacks (15) of n=6 individuals using ImageJ. Central band of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically different groups (P < 0.05) as determined by one-way ANOVA. The experiment was repeated twice with similar results.


( B ) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N . benthamiana plants at 1 and 2dpi . Agrobacterium - mediated transient expression of AIMp - RFP serves as a control for autophagy suppression . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results .

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(B) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N. benthamiana plants at 1 and 2dpi. Agrobacterium-mediated transient expression of AIMp-RFP serves as a control for autophagy suppression. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( C ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ was co - infiltrated with Agrobacteria containing the luciferase reporter constructs . Coexpression of RFP - AIMp serves as a control for autophagy inhibition . Expression of the latter was confirmed with western blot ( inset ) . Renilla ( RLUC ) and Firefly ( FLUC ) luciferase activities were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . The experiment was repeated more than 3 times with similar results .

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(C) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ was co-infiltrated with Agrobacteria containing the luciferase reporter constructs. Coexpression of RFP-AIMp serves as a control for autophagy inhibition. Expression of the latter was confirmed with western blot (inset). Renilla (RLUC) and Firefly (FLUC) luciferase activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P<0.001) was revealed by Student's t-test. The experiment was repeated more than 3 times with similar results.


( D ) RT - qPCR analysis of NbATG8 - 1 . 1 / 1 . 2 , NbATG8 - 2 . 1 / 2 . 2 and NbJoka2 transcript levels upon challenge of N . benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants . Values represent expression relative to mock control of respective time point and were normalized to actin . Values are biological replicates ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) was revealed by Student ' s t - test .

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(D) RT-qPCR analysis of NbATG8-1.1/1.2, NbATG8-2.1/2.2 and NbJoka2 transcript levels upon challenge of N. benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants. Values represent expression relative to mock control of respective time point and were normalized to actin. Values are biological replicates (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (*P < 0.05, **P < 0.01, *** P < 0.001) was revealed by Student's t-test.


( E ) Bacterial density in leaves of N . benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp - RFP . Leaves were syringe - infiltrated with OD600 = 0 . 0004 , and colony - forming units were counted at 6 dpi . Compared to empty vector control ( EV ) , AIMp expressing plants ( n = 6 ) harbour significantly more bacteria . Bacterial growth was repeated with the same result in 12 plants over two independent experiments . Red and yellow data points indicate independent repeats of the experiment . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . Expression of RFP - AIMp was verified at 6 dpi with an anti - RFP blot ( inset ) .

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(E) Bacterial density in leaves of N. benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp-RFP. Leaves were syringe-infiltrated with OD600 = 0.0004, and colony-forming units were counted at 6 dpi. Compared to empty vector control (EV), AIMp expressing plants (n=6) harbour significantly more bacteria. Bacterial growth was repeated with the same result in 12 plants over two independent experiments. Red and yellow data points indicate independent repeats of the experiment. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P < 0.001) was revealed by Student's t-test. Expression of RFP-AIMp was verified at 6 dpi with an anti-RFP blot (inset).


( A ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . XopL or GFP constructs were co - infiltrated . RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities to the mean of control ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( P < 0 . 01 ) was shown by Student ' s t - test . The experiment was repeated more than 3 times by with similar results .

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(A) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. XopL or GFP constructs were co-infiltrated. RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities to the mean of control (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (P < 0.01) was shown by Student's t-test. The experiment was repeated more than 3 times by with similar results.


( B ) Immunoblot analysis of NBR1 and ATG8 protein levels in N . benthamiana plants transiently expressing GFP - XopL or GFP control at 2dpi verified with an anti - GFP antibody ( same blot was split for visualization purpose ) . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated at least three times with similar results .

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(B) Immunoblot analysis of NBR1 and ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL or GFP control at 2dpi verified with an anti-GFP antibody (same blot was split for visualization purpose). Ponceau Staining (PS) served as a loading control. The experiment was repeated at least three times with similar results.