Figure 3 ( A - E ) Representative live cell microscopy images of D . discoideum infected with PflaA - GFP - producing L . pneumophila . D . discoideum Ax3 producing cytosolic mCherry ( pDM1042 ) was infected ( MOI 5 ) with ( A , B ) wild - type L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) , ( C , D ) JR32 ( Ptac - mCerulean - PflaA - gfp ) or ( E ) ∆ flaA ( Ptac - mCerulean - PflaA - gfp ) and embedded in agarose for 48 h . PflaA - GFP - positive L . pneumophila ( A ) escapes the LCV ( 0 sec ) , followed by other bacteria , which are contained in the cytosol and localize to the plasma membrane , where a PflaA - GFP - positive L . pneumophila first leaves the ruptured host cell ( 185 sec ) , or ( B ) triggers host cell death ( loss of cytosolic mCherry ) upon exit of the amoeba . PflaA - GFP - positive L . pneumophila ( C ) escape the LCV after 75 sec and spread within the cytosol for several minutes , and ( D ) are the first to exit the host cell , leading to the loss of cytosolic mCherry and host cell death . ( E ) L . pneumophila ∆ flaA lyses the LCV and host cell less vigorously . Scale bars : 5 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-E) Representative live cell microscopy images of D. discoideum infected with PflaA-GFP-producing L. pneumophila. D. discoideum Ax3 producing cytosolic mCherry (pDM1042) was infected (MOI 5) with (A, B) wild-type L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp), (C, D) JR32 (Ptac-mCerulean-PflaA-gfp) or (E) ∆flaA (Ptac-mCerulean-PflaA-gfp) and embedded in agarose for 48 h. PflaA-GFP-positive L. pneumophila (A) escapes the LCV (0 sec), followed by other bacteria, which are contained in the cytosol and localize to the plasma membrane, where a PflaA-GFP-positive L. pneumophila first leaves the ruptured host cell (185 sec), or (B) triggers host cell death (loss of cytosolic mCherry) upon exit of the amoeba. PflaA-GFP-positive L. pneumophila (C) escape the LCV after 75 sec and spread within the cytosol for several minutes, and (D) are the first to exit the host cell, leading to the loss of cytosolic mCherry and host cell death. (E) L. pneumophila ∆flaA lyses the LCV and host cell less vigorously. Scale bars: 5 µm.


Figure 4 ( B ) L . pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae . A . castellanii amoebae were infected ( MOI 5 , 52 h ) with L . pneumophila JR32 , ∆ flaA or ∆ lqsA harboring Ptac - mCherry - PflaA - gfp and embedded in 0 . 5 % agarose / PYG medium . The fluorescence images are representative for the L . pneumophila strains indicated : JR32 ( n = 25 ) , ∆ flaA ( n = 13 ) or ∆ lqsA ( n = 19 ) . Scale bars : 20 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) L. pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae. A. castellanii amoebae were infected (MOI 5, 52 h) with L. pneumophila JR32, ∆flaA or ∆lqsA harboring Ptac-mCherry-PflaA-gfp and embedded in 0.5% agarose/PYG medium. The fluorescence images are representative for the L. pneumophila strains indicated: JR32 (n = 25), ∆flaA (n = 13) or ∆lqsA (n = 19). Scale bars: 20 µm.


Figure 5 ( A , B ) Role of the Lqs system for PflaA expression in intracellular L . pneumophila . A . castellanii was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , and ( A ) fixed and analyzed by confocal microscopy ( representative 3D reconstructions , scale bars : 10 µm ) , or ( B ) lysed , fixed and analyzed by flow cytometry . ( C - F ) Role of the Lqs system for LCV escape and cytosolic localization of L . pneumophila . ( C , D ) A . castellanii or ( E , F ) D . discoideum was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , fixed and treated with an anti ‐ ubiquitin antibody . Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy . Representative images of ( C ) A . castellanii ( scale bars : 10 µm ) or ( E ) D . discoideum ( scale bars : 5 μm ) infected with L . pneumophila JR32 escaping or not from the LCV are shown . The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells : ( D ) JR32 : n = 482 ; ΔlqsA : n = 834 ; ΔlqsR : n = 572 ; ΔlqsS : n = 729 ; ΔlqsT : n = 1469 ; ΔlqsS - ΔlqsT : n = 693 ; ( F ) : JR32 : n = 136 ; ΔlqsA : n = 201 ; ΔlqsR : n = 303 ; ΔlqsS : n = 183 ; ΔlqsT : n = 534 ; ΔlqsS - ΔlqsT : n = 180 . ( G , H ) D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - GFP and cytosolic - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , ∆ lqsS - ∆ lqsT , or ∆ flaA harboring Ptac - mCerulean . Representative images of ( G ) D . discoideum infected with L . - pneumophila JR32 in intact cells with intact LCV ( 18 . 5 % ) , lysed cells and lysed LCVs ( 60 . 2 % ) , intact cells with lysed LCVs ( 15 . 5 % ) and lysed cells with intact LCVS ( 5 . 8 % ) are shown ( scale bars : 5 µm ) . ( H ) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane : JR32 : n = 426 ; ΔlqsA : n = 468 ; ΔlqsR : n = 344 ; ΔlqsS : n = 325 ; ΔlqsT : n = 287 ; ΔlqsS - ΔlqsT : n = 289 ; ΔflaA : n = 248 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A, B) Role of the Lqs system for PflaA expression in intracellular L. pneumophila. A. castellanii was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, and (A) fixed and analyzed by confocal microscopy (representative 3D reconstructions, scale bars: 10 µm), or (B) lysed, fixed and analyzed by flow cytometry.(C-F) Role of the Lqs system for LCV escape and cytosolic localization of L. pneumophila. (C, D) A. castellanii or (E, F) D. discoideum was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, fixed and treated with an anti‐ubiquitin antibody. Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy. Representative images of (C) A. castellanii (scale bars: 10 µm) or (E) D. discoideum (scale bars: 5 μm) infected with L. pneumophila JR32 escaping or not from the LCV are shown. The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells: (D) JR32: n = 482; ΔlqsA: n = 834; ΔlqsR: n = 572; ΔlqsS: n = 729; ΔlqsT: n = 1469; ΔlqsS-ΔlqsT: n = 693; (F): JR32: n = 136; ΔlqsA: n = 201; ΔlqsR: n = 303; ΔlqsS: n = 183; ΔlqsT: n = 534; ΔlqsS-ΔlqsT: n = 180.(G, H) D. discoideum producing the LCV/PtdIns(4)P probe P4C-GFP and cytosolic-mCherry was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, ∆lqsS-∆lqsT, or ∆flaA harboring Ptac-mCerulean. Representative images of (G) D. discoideum infected with L.-pneumophila JR32 in intact cells with intact LCV (18.5%), lysed cells and lysed LCVs (60.2%), intact cells with lysed LCVs (15.5%) and lysed cells with intact LCVS (5.8%) are shown (scale bars: 5 µm). (H) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane: JR32: n = 426; ΔlqsA: n = 468; ΔlqsR: n = 344; ΔlqsS: n = 325; ΔlqsT: n = 287; ΔlqsS-ΔlqsT: n = 289; ΔflaA: n = 248.


Figure 6D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila wild - type strains ( JR32 , Corby ) , or phospholipase triple mutants ( ∆ plcABC , ∆ plaACD ) harboring the Ptac - mCerulean - PflaA - gfp reporter construct , and fixed and treated with an anti ‐ ubiquitin antibody , LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact , P4C - mCherry - positive LCVs or ubiquitin - decorated , cytosolic bacteria . ( A ) Representative images of amoebae harboring L . pneumophila confined within LCVs or released from the LCV to the cytosol are shown ( scale bars : 5 μm ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and fixed and treated with an anti‐ubiquitin antibody, LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (A) Representative images of amoebae harboring L. pneumophila confined within LCVs or released from the LCV to the cytosol are shown (scale bars: 5 μm).


Figure 2 ( A ) Pancreatic cancer cells ( PANC - 1 ) and normal human fibroblasts ( NHFs ) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points , cells were incubated with WST - 1 cell viability dye for 4 hours . Absorbance was read at 450 nm and results were expressed as a percent of the control . Values are expressed as mean ± SD from quadruplicates ; ( i ) PANC - 1 treated with NSC 51046 ( Interaction between each concentration : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( ii ) NHF treated with NSC 51046 ( Interaction between each concentration : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iii ) PANC - 1 treated with Green 1 ( Interaction between each concentration : p = 0 . 9955 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iv ) NHF treated with Green 1 ( Interaction between each concentration : p = 0 . 3642 ; Control versus column factor : * p = 0 . 0370 ) ( B ) Following treatment of human acute T cell leukemia cells ( Jurkat ) with Green 1 , a trypan blue excluding assay was carried out . Cells were stained with trypan blue cell membrane impermeable dye and the numbers of trypan blue positive cells were obtained using a hemocytometer . Results are expressed as percent of trypan blue positive cells , indicating dead cells with non - intact cell membranes . Values are expressed as mean ± SD from two independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Pancreatic cancer cells (PANC-1) and normal human fibroblasts (NHFs) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points, cells were incubated with WST-1 cell viability dye for 4 hours. Absorbance was read at 450 nm and results were expressed as a percent of the control. Values are expressed as mean ± SD from quadruplicates; (i) PANC-1 treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (ii) NHF treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (iii) PANC-1 treated with Green 1 (Interaction between each concentration: p = 0.9955; Control versus column factor: ****p<0.0001); (iv) NHF treated with Green 1 (Interaction between each concentration: p = 0.3642; Control versus column factor: *p = 0.0370)(B) Following treatment of human acute T cell leukemia cells (Jurkat) with Green 1, a trypan blue excluding assay was carried out. Cells were stained with trypan blue cell membrane impermeable dye and the numbers of trypan blue positive cells were obtained using a hemocytometer. Results are expressed as percent of trypan blue positive cells, indicating dead cells with non-intact cell membranes. Values are expressed as mean ± SD from two independent experiments.


Figure 3Following treatment with either Green 1 ( A ) Pancreatic cancer cells ( BxPC - 3 and PANC - 1 ) and normal human and fetal fibroblasts ( NHFs and NFFs ) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis . Brightly stained , condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis . ( B ) PANC - 1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046 . Cells were counterstained with Hoechst 33342 dye and propidium iodide ( PI ) to visualize nuclear morphology and cell death . Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Following treatment with either Green 1(A) Pancreatic cancer cells (BxPC-3 and PANC-1) and normal human and fetal fibroblasts (NHFs and NFFs) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis. Brightly stained, condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis.(B) PANC-1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046. Cells were counterstained with Hoechst 33342 dye and propidium iodide (PI) to visualize nuclear morphology and cell death. Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine.


Figure 4 ( A ) Pancreatic cancer cells , human leukemia cells ( Jurkat ) and NHFs were incubated with Monodansylcadaverine ( MDC ) to stain autophagic vacuoles and propidium iodide ( PI ) for cell death to determine if autophagic induction led to cell death . Bright , punctate staining is indicative of autophagy , as seen in our positive control ( Tamoxifen treated cells ) . Images were taken at 400X magnification on a fluorescent microscope . ( B ) Western blot analyses of Beclin - 1 expression and LC3 conversion were conducted on PANC - 1 cells treated with 5 . 0 µM Green 1 at various time points . β - actin was measured as an internal control . Samples treated for 3 and 6 hours were compared to the early control , while the sample treated for 48 hours was compared to the 48 - hour control . Results are expressed as mean ± SD from two independent experiments for both Western blots .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Pancreatic cancer cells, human leukemia cells (Jurkat) and NHFs were incubated with Monodansylcadaverine (MDC) to stain autophagic vacuoles and propidium iodide (PI) for cell death to determine if autophagic induction led to cell death. Bright, punctate staining is indicative of autophagy, as seen in our positive control (Tamoxifen treated cells). Images were taken at 400X magnification on a fluorescent microscope.(B) Western blot analyses of Beclin-1 expression and LC3 conversion were conducted on PANC-1 cells treated with 5.0 µM Green 1 at various time points. β-actin was measured as an internal control. Samples treated for 3 and 6 hours were compared to the early control, while the sample treated for 48 hours was compared to the 48-hour control. Results are expressed as mean ± SD from two independent experiments for both Western blots.


Figure 5 ( A ) Colchicine derivatives ( Green 1 and NSC 51046 ) were incubated with porcine tubulin for an hour in a 96 - well plate . The absorbance ( OD 340 nm ) was obtained every minute during the one hour incubation . Colchicine was used for comparison to the derivatives . ( B ) Isolated mitochondria from PANC - 1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase ( HRP ) . Results were compared to control untreated mitochondria , colchicine treated mitochondria and positive control , paraquat ( PQ ) . Fluorescence readings were taken in 5 min intervals for 4 hours at Ex . 560 nm and Em . 590 nm and expressed as relative fluorescence units ( RFU ) . Results are a representative of three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Colchicine derivatives (Green 1 and NSC 51046) were incubated with porcine tubulin for an hour in a 96-well plate. The absorbance (OD 340 nm) was obtained every minute during the one hour incubation. Colchicine was used for comparison to the derivatives.(B) Isolated mitochondria from PANC-1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase (HRP). Results were compared to control untreated mitochondria, colchicine treated mitochondria and positive control, paraquat (PQ). Fluorescence readings were taken in 5 min intervals for 4 hours at Ex. 560 nm and Em.590 nm and expressed as relative fluorescence units (RFU). Results are a representative of three independent experiments.


Figure 6Following acclimatization , CD - 1 nu / nu mice were separated into two groups , one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS ( 10 µL in 200 µL PBS ) , while the second group received subcutaneous injections of Green 1 ( 10 mg / kg / day for a total volume of 10 µL Green 1 / 200 µL PBS ) three times a week for a period of one month . ( A ) Mice weights were recorded twice a week for the duration of the study . ( B ) Following the study , mice were sacrificed and their organs were obtained for histopathological assessment . Hematoxylin and Eosin staining was carried out on the tissue samples .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Following acclimatization, CD-1 nu/nu mice were separated into two groups, one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS (10 µL in 200 µL PBS), while the second group received subcutaneous injections of Green 1 (10 mg/kg/day for a total volume of 10 µL Green 1/200 µL PBS) three times a week for a period of one month. (A) Mice weights were recorded twice a week for the duration of the study.(B) Following the study, mice were sacrificed and their organs were obtained for histopathological assessment. Hematoxylin and Eosin staining was carried out on the tissue samples.


Figure 2 , Scaled reduced cost analysis for uptake rates of amino acids at different growth rates . Colour represents the scaled reduced cost value . A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates. Colour represents the scaled reduced cost value. A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate.


Figure 4 , Protein fold changes of the CcpA mutant over wild type . Log2 fold changes were calculated from LFQ values p values were calculated based on 2 - sided two sample t - tests with a FDR threshold of 0 . 05 and S 0 equal to 1 ( Tusher et al , 2001 ) . Proteins outside the significance lines ( FDR = 0 . 05 , S 0 = 1 ) are significantly changed in expression . Example sets of proteins are highlighted and are labeled with their gene names .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4, Protein fold changes of the CcpA mutant over wild type. Log2 fold changes were calculated from LFQ values p values were calculated based on 2-sided two sample t-tests with a FDR threshold of 0.05 and S0 equal to 1 (Tusher et al, 2001). Proteins outside the significance lines (FDR = 0.05, S0 = 1) are significantly changed in expression. Example sets of proteins are highlighted and are labeled with their gene names.


Figure 2 ( C ) Change in the expression levels of 36 genes defining the host - targeted anti - viral signature . log2 fold change at 24 hours post SARS - CoV - 2 infection from A549 cells are shown . ( D ) Change in the expression levels of 17 genes defining anti - cytokine signature ; log2 fold change at 24 hours post high SARS - CoV - 2 infection from A549 - ACE2 cells are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) Change in the expression levels of 36 genes defining the host-targeted anti-viral signature. log2 fold change at 24 hours post SARS-CoV-2 infection from A549 cells are shown.(D) Change in the expression levels of 17 genes defining anti-cytokine signature; log2 fold change at 24 hours post high SARS-CoV-2 infection from A549-ACE2 cells are shown.


Figure 5 ( A ) Representative fluorescence images of Mock , SARS - CoV - 2 infected ( Ctrl ) , and Salmeterol - treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress . Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments : Segmentation images show how cells were segmented and identified as spike positive . Purple , nuclei ; cyan , spike . Scale bar , 100 μm . ( B ) Violin plots of Vero - E6 cells labeled for Spike protein . The Multi - wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell . The plots show the population distribution of the integrated signal for all of the treatments . The Boxes in the plot show the interquartile range ( IQR ) with the top and bottom edges marking the 75th and 25th percentiles , respectively . The horizontal line in the box is the median value , and the whiskers are defined to be 1 . 5 IQR . The ordinate is a log scale . The effect of the treatment is assessed quantitatively by changes in the median signal level , and qualitatively by observing changes in the modes . The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut - off to quantify the number of cells that are positive or negative for the Spike signal . The statistics table below the plots shows the number of cells counted in each treatment group , and the median of the population .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.(B) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population.


Figure 6HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates , pretreated with 7 - point gradients of test compounds for 1 - 2 h , and co - cultured for 4 hours with HEK293 donor cells expressing SARS - CoV - 2 spike and GFP , or donor cells expressing GFP only ( no spike ) . Images of GFP - positive objects were acquired on a confocal high - content imager and analyzed for syncytia formation and integrated GFP area ( total GFP ) as a measure of cytotoxicity , using a CNT algorithm as described in the Materials and Methods . Data are the aggregate of 8 independent biological repeats ; where errors are shown they represent SD from matching concentrations in at least three experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates, pretreated with 7-point gradients of test compounds for 1-2 h, and co-cultured for 4 hours with HEK293 donor cells expressing SARS-CoV-2 spike and GFP, or donor cells expressing GFP only (no spike). Images of GFP-positive objects were acquired on a confocal high-content imager and analyzed for syncytia formation and integrated GFP area (total GFP) as a measure of cytotoxicity, using a CNT algorithm as described in the Materials and Methods. Data are the aggregate of 8 independent biological repeats; where errors are shown they represent SD from matching concentrations in at least three experiments.


Figure 1V . cholerae wild - type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase ( OD600 of 1 . 5 ) and induced with L - arabinose ( 0 . 2 % final conc . ) . Expression of MicV and VrrA was monitored on Northern blots . 5S rRNA served as loading control . V . cholerae wild - type , ΔvchM and ΔvchM ΔrpoE strains harboring PmicV : : mKate2 plasmids were grown in M9 medium . Samples were collected during various growth phases and analyzed for fluorescence . V . cholerae wild - type and ΔrpoE strains carrying PmicV : : mKate2 plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 . V . cholerae wild - type and ΔrpoE strains carrying PvrrA : : mKate2 ( E ) plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 . V . cholerae wild - type , ΔmicV , ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.V. cholerae wild-type, ΔvchM and ΔvchM ΔrpoE strains harboring PmicV::mKate2 plasmids were grown in M9 medium. Samples were collected during various growth phases and analyzed for fluorescence.V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.V. cholerae wild-type and ΔrpoE strains carrying PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


Figure 2V . cholerae ΔvrrA ΔmicV strains carrying pBAD - micV , pBAD - vrrA or an empty vector control ( pCtr ) were cultivated to early stationary phase ( OD600 of 1 . 5 ) in LB . Cells were treated with L - arabinose ( 0 . 2 % final conc . ) and RNA samples were collected at the indicated time points after induction . Northern blot analysis was performed to determine VrrA , MicV and ompT levels . 5S rRNA served as loading control . For comparison , RNA samples of a wild - type strain carrying pCtr were collected during various growth phases , which indicated ~ 18 - fold and ~ 7 - fold higher levels of VrrA and MicV expressed from the pBAD plasmids , respectively ( see Source data for quantifications ) . V . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to ( B ) : regulated by both sRNAsV . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to regulated only by MicVV . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to regulated only by

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to (B): regulated by both sRNAsV. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by MicVV. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by VrrA


Figure 3V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : gfp or ompT M1 * : : gfp fusions ( D ) , ushA : : gfp or ushA M1 * : : gfp fusions ( E ) , or lpp : : gfp or lpp M2 * : : gfp fusions ( F ) and an empty vector control ( pCtr ) , the micV expression plasmids ( pMicV , pMicV M1 ) or the vrrA expression plasmids ( pVrrA , pVrrA M2 ) were grown in M9 and GFP fluorescence was measured . M1 and M2 denote the mutations indicated in ( A , B , C ) . Fluorescence of the control strains was set to 1 . V . cholerae ΔrpoE , ΔrpoE ΔvrrA , ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD - rpoE or an empty vector control ( pCtr ) were grown to OD600 of 1 . 5 and L - arabinose ( 0 . 2 % final conc . ) was added . RNA samples were collected at the indicated time points and monitored for ompT ( G ) , ushA ( H ) , or lpp ( I ) levels using qRT - PCR .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3V. cholerae ΔvrrA ΔmicV strains carrying the ompT::gfp or ompT M1*::gfp fusions (D), ushA::gfp or ushA M1*::gfp fusions (E), or lpp::gfp or lpp M2*::gfp fusions (F) and an empty vector control (pCtr), the micV expression plasmids (pMicV, pMicV M1) or the vrrA expression plasmids (pVrrA, pVrrA M2) were grown in M9 and GFP fluorescence was measured. M1 and M2 denote the mutations indicated in (A, B, C). Fluorescence of the control strains was set to 1.V. cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD-rpoE or an empty vector control (pCtr) were grown to OD600 of 1.5 and L-arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT-PCR.


Figure 4V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : 3XFLAG gene and pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and analyzed for MicV , VrrA and RybB expression on Northern blots . OmpT : : 3xFLAG production was tested on Western blots . RNAPα and 5S rRNA served as loading controls for Western and Northern blots , respectively . E . coli wild - type strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and investigated on Northern blots and SDS - PAGE , respectively . For comparison , we included the E . coli insertional mutant strains ompA : : kanR and ompC : : kanR for specific assignment of OmpA and OmpC bands . V . cholerae wild - type and ΔrpoE strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4V. cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to an OD600 of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.E. coli wild-type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS-PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kanR and ompC::kanR for specific assignment of OmpA and OmpC bands.V. cholerae wild-type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


Figure 5V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library after consecutive selection experiments ( Sel1 , Sel2 , Sel3 ) were grown in LB to OD600 of 0 . 2 . Cells were treated with ethanol ( 3 . 5 % final conc . ) for 6 hours . Serial dilutions were prepared and spotted onto agar plates . R1 and R2 indicate two independent biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.


Figure 6V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library before ( input ) or after consecutive ethanol selection experiments ( Sel1 , Sel2 , Sel3 ) were cultivated in LB to OD600 of 2 . 0 . Membrane fractions were analyzed by SDS - PAGE . The indicated bands were analyzed by mass spectrometry . V . cholerae ΔvrrA ΔmicV cells expressing the ompA : : 3xFLAG gene and carrying an empty vector control ( pCtr ) , or plasmids producing the 15 most highly enriched sRNA variants ( sRNA variants 1 - 15 ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and tested for sRNA and OmpA : : 3xFLAG expression on Northern and Western blots , respectively ( with RNAPα and 5S rRNA as loading controls ) . V . cholerae ΔvrrA ΔmicV strains carrying the ompA : : gfp fusion and an empty vector control ( pCtr ) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . V . cholerae wild - type , ΔrpoE , ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, Sel3) were cultivated in LB to OD600 of 2.0. Membrane fractions were analyzed by SDS-PAGE. The indicated bands were analyzed by mass spectrometry.V. cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1-15) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with RNAPα and 5S rRNA as loading controls).V. cholerae ΔvrrA ΔmicV strains carrying the ompA::gfp fusion and an empty vector control (pCtr) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1.V. cholerae wild-type, ΔrpoE, ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


Figure 1 ( A ) Immunoblot showing the activation of PI3K / AKT pathway ( by assessing the levels of p - AKT Ser473 ) of ECs 24h after induction of Pik3caH1047R expression . ( B ) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression . Differentially expressed genes ( DEGs ) ( p adj < 0 . 05 ) in Pik3caH1047R over Pik3caWT ECs at 24h ( grey , unchanged ; orange , downregulated ; purple , upregulated ) . n = 4 biological replicates per genotype . ( D ) Immunoblot showing the activation of PI3K / AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation ( - ) and 30 min stimulation with 20 % fetal bovine serum ( FBS ) ( + ) . ( E , F ) ( E ) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum ( - ) or 20 % serum ( + ) for 24h . ( F ) Percentage of proliferative ECs ( in S and G2M phases ) from Fig 1E . Error bars are s . e . m . n ≥ 3 biological replicates per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 and * * * * p < 0 . 0001 were considered statistically significant .

O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Immunoblot showing the activation of PI3K/AKT pathway (by assessing the levels of p-AKT Ser473) of ECs 24h after induction of Pik3caH1047R expression.(B) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression. Differentially expressed genes (DEGs) (p adj < 0.05) in Pik3caH1047R over Pik3caWT ECs at 24h (grey, unchanged; orange, downregulated; purple, upregulated). n=4 biological replicates per genotype.(D) Immunoblot showing the activation of PI3K/AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation (-) and 30 min stimulation with 20% fetal bovine serum (FBS) (+).(E, F) (E) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum (-) or 20% serum (+) for 24h. (F) Percentage of proliferative ECs (in S and G2M phases) from Fig 1E. Error bars are s.e.m. n ≥ 3 biological replicates per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 and ****p < 0.0001 were considered statistically significant.


Figure 2 ( B ) Representative images of control and EC - Pik3caH1047R / WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels . Scale bars : 150 μm . ( D ) Quantification of IB4 - positive areas per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype . ( E ) Representative images of EC - mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP . Scale bars : 150 μm . ( F ) Quantification of GFP / IB4 ratio per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Representative images of control and EC-Pik3caH1047R/WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels. Scale bars: 150 μm.(D) Quantification of IB4-positive areas per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype.(E) Representative images of EC-mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP. Scale bars: 150 μm. (F) Quantification of GFP/IB4 ratio per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


Figure 3 ( A , B ) Representative images of EC - Pik3caH1047R / WT ( A ) and EC - mTmG ( B ) P6 retinas from mice treated with decreasing doses of 4 - OHT on P1 . Retinas were stained for blood vessels ( IB4 ) and GFP as indicated . Scale bars : 150 μm . ( C ) Quantification of GFP / IB4 ratio of EC - mTmG retinas . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant . ( D ) Quantification of IB4 - positive area per retina in Pik3caH1047R / WT retinas . Data presented as a percentage of the control for each 4 - OHT dose . Error bars are s . e . m . n = 4 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice treated with decreasing doses of 4-OHT on P1. Retinas were stained for blood vessels (IB4) and GFP as indicated. Scale bars: 150 μm.(C) Quantification of GFP/IB4 ratio of EC-mTmG retinas. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.(D) Quantification of IB4-positive area per retina in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each 4-OHT dose. Error bars are s.e.m. n = 4 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


Figure 4 ( A ) Representative images of EC - Pik3caH1047R / WT P6 retinas isolated from mice treated with 0 . 125 mg / kg of 4 - OHT on P1 . Retinas were immunostained for blood vessels ( IB4 ) . Red asterisks show arteries and arterioles and yellow arrowheads veins . Scale bars : 150 μm ( left panel ) and 30 μm ( right , high magnification panels ) . ( B ) Representative images of P6 retinas from control and EC - Pik3caH1047R / WT mice treated with 0 . 125 mg / kg 4 - OHT on P1 , following immunostaining for p - S6 ( S235 / 236 ) and blood vessels ( IB4 ) . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( C ) Representative control and EC - Pik3caH1047R / WT P6 retinas immunostained for blood vessels ( IB4 ) , EC nuclei ( Erg ) and EdU . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( D - G ) Quantification of ( D ) p - S6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) , ( E ) EC proliferation by EdU staining , ( F ) EC number by Erg positive cells and ( G ) retinal vascularity by IB4 - positive area in control and EC - Pik3caH1047R / WT retinas . Error bars are s . e . m . n > 5 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 was considered statistically significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels). (B) Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/236) and blood vessels (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (D-G) Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.


Figure 5 ( B , C ) Representative images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panel ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B, C) Representative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


Figure 6 ( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 3 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 3 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


Figure 7 ( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 were considered statistically significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.


Figure 8 ( B ) Representative sequencing chromatograms of PIK3CA and TEK mutant VM - derived ECs . Arrows show the detected point mutations . ( C ) Representative confocal images of PIK3CA and TEK patient - derived ECs immunostained for VE - cadherin ( EC - specific junctional protein ) and ERG ( EC - specific transcription factor ) . Cell nuclei were visualised with DAPI . Scale bars : 30 μm . ( D ) Immunoblot showing the activation of PI3K / AKT / mTORC1 pathway ( by assessing the levels of p - AKT and p - S6 ) among different PIK3CA and TEK patient - derived ECs . Primary HUVEC were used as wild - type control . ( E - F ) Immunoblot showing the impact of miransertib at increasing doses on PI3K / AKT / mTORC1 pathway ( by assessing p - AKT and p - S6 levels ) in ( E ) PIK3CA and ( F ) TEK mutant patient - derived ECs . ( G ) Wild - type ( HUVECs ) , PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay . Fitting curves and 95 % CI IC50 values for wild - type ( HUVECs ) , PIK3CA and TEK ECs are shown . For PIK3CA and TEK - mutant ECs n = 3 biological replicates ( patients ) ; for wild - type ECs ( HUVECs ) n = 4 technical replicates PIK3CA - mutant vs . TEK - mutant p = 0 . 501 ; PIK3CA - mutant vs . HUVECs p = 0 . 0815 ; TEK - mutant vs . HUVECs : p = 0 . 4085 . Statistical analysis was performed by comparison of best - fit values using the extra sum - of - squares F test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(B) Representative sequencing chromatograms of PIK3CA and TEK mutant VM-derived ECs. Arrows show the detected point mutations.(C) Representative confocal images of PIK3CA and TEK patient-derived ECs immunostained for VE-cadherin (EC-specific junctional protein) and ERG (EC-specific transcription factor). Cell nuclei were visualised with DAPI. Scale bars: 30 μm.(D) Immunoblot showing the activation of PI3K/AKT/mTORC1 pathway (by assessing the levels of p-AKT and p-S6) among different PIK3CA and TEK patient-derived ECs. Primary HUVEC were used as wild-type control.(E-F) Immunoblot showing the impact of miransertib at increasing doses on PI3K/AKT/mTORC1 pathway (by assessing p-AKT and p-S6 levels) in (E) PIK3CA and (F) TEK mutant patient-derived ECs.(G) Wild-type (HUVECs), PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50 values for wild-type (HUVECs), PIK3CA and TEK ECs are shown. For PIK3CA and TEK-mutant ECs n = 3 biological replicates (patients); for wild-type ECs (HUVECs) n = 4 technical replicates PIK3CA-mutant vs. TEK-mutant p = 0.501; PIK3CA-mutant vs. HUVECs p = 0.0815; TEK-mutant vs. HUVECs: p = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test.


Figure 1Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL / 6 mouse lines . Tissues were isolated from indicated C57BL / 6 or iNKT TN mouse lines and stained with anti - CD3 and CD1d ( PBS57 ) tetramer . Spleen , mLN , sdLN , liver , adipose , and lung lymphocytes from Jα18 - / - or Vα14 mice were stimulated in vitro with RAW - CD1d cells and 1μg α - GalCer . An additional sample of Vα14 lymphocytes from each organ was plated with RAW - CD1d cells but no α - GalCer . Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array . Error bars are SD of mean values from three different mice per group . Results shown are representative of two independent experiments where n = 3 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d (PBS57) tetramer.Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18-/- or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n=3 biological replicates.


Figure 2Lymphocytes from the indicated tissues of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer , before they were fixed , permeabilized , and stained with antibodies to T - bet , RORγt , and PLZF . Results shown are gated on CD3 + CD1d - tetramer + cells . The percentage of CD3 + CD1d - tetramer + iNKT cells in each organ that stained positively for PLZF , T - bet , and RORγt are shown . * * p < 0 . 01 , Mann - Whitney test . Error bars are SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells.The percentage of CD3+ CD1d-tetramer+ iNKT cells in each organ that stained positively for PLZF, T-bet, and RORγt are shown. **p<0.01, Mann-Whitney test. Error bars are SD.


Figure 3Lymphocytes from the Peyer ' s patches of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer . Percentage of lymphocytes that were CD3 + CD1d - tetramer + iNKT cells among the Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 22 ; Vα14 n = 16 ; Jα18 - / - n = 8 . ( C - E ) Mice were analyzed for total fecal IgA by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 . ( C - E ) Mice were analyzed for total fecal IgG1 ( D ) or total serum IgG1 ( E ) by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 . Percentages of total B cells that were IgG1 + in the spleen , mLN , and Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 15 ; Vα14 n = 15 ; Jα18 - / - n =

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Lymphocytes from the Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer. Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among the Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=22; Vα14 n=16; Jα18-/- n=8.(C-E)Mice were analyzed for total fecal IgA by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.(C-E)Mice were analyzed for total fecal IgG1 (D) or total serum IgG1 (E) by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.Percentages of total B cells that were IgG1+ in the spleen, mLN, and Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=15; Vα14 n=15; Jα18-/- n=5


Figure 4CD1d ( PBS57 ) tetramer + CD3 + cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4 + CD3 + CD1d tetramer - cells from PP ( PP CD4 ) . RNAseq was performed . Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample . Spleen , mLN , and PP lymphocytes from Vα14 and C57BL / 6 mice were stimulated with PMA and ionomycin . Lymphocytes were stained with anti - CD3 , CD1d ( PBS57 ) tetramer , anti - IL - 4 , and anti - IFNγ . Percentages of iNKT cells and non - iNKT T cells within the population of CD3 + IL - 4 + cells and CD3 + IFNγ + cells , n = 5 mice for C57BL / 6 group and n = 4 mice for Vα14 group . Error bars are SD . Vα14 TN mice were administered α - GalCer either 2μg intravenously or 5μg by oral gavage . Mice were given Brefeldin A intraperitoneally after 30 min , and tissues were harvested 3 hours later . Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with antibodies to IL - 4 , IFNγ , and IL - 17 . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells . Quantification of data from C , n = 2 mice per group . Mann - Whitney test . Error bars are SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample.Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD.Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.Quantification of data from C, n=2 mice per group. Mann-Whitney test. Error bars are SEM.


Figure 5Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α - GalCer and with or without agnostic anti - CD40 . IgM was measured by ELISA of culture supernatants 4 days later . Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti - CD40 activated wild type B cells with or without 1μg α - GalCer and with blocking antibody to CD1d ( 1μg / mL , clone 1B1 ) or isotype control as indicated . IgM , IgG1 , and IgA were measured by ELISA of 4 day culture supernatants . Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . IL - 4 were measured by ELISA of culture supernatants . Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 ( D - E ) were measured by ELISA of culture supernatants . Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + and IgA + class switched B cells were enumerated by flow cytometry after 4 days of cocultureSpleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + class switched B cells were enumerated by flow cytometry after 4 days of

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants.Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. IL-4 were measured by ELISA of culture supernatants.Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1 (D-E) were measured by ELISA of culture supernatants.Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of cocultureSpleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


Figure 6OVA - specific antibody titers from feces were determined by ELISA from the mice in ( A ) . Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD . OVA - specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD . Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with the indicated antibodies . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells . Quantification of data from E , n = 3 mice per group . Mann - Whitney test . Error bars are SEM . * p < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6OVA-specific antibody titers from feces were determined by ELISA from the mice in (A). Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.OVA-specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with the indicated antibodies. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. Quantification of data from E, n=3 mice per group. Mann-Whitney test. Error bars are SEM. *p<0.05.


Figure 7Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP - iNKT cells . Heatmap of FPKM values from genes uniquely upregulated in PP - iNKT cells as compared to both spleen iNKT and PP CD4 cells . Red indicates higher expression and blue indicates lower expression . Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies , analyzed by flow cytometry and gated on CD1d ( PBS57 ) tetramer + CD3 + cells . Quantification of data from D , n = 11 mice per group . Mann - Whitney test . Error bars are SEM .

O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 7Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP-iNKT cells.Heatmap of FPKM values from genes uniquely upregulated in PP-iNKT cells as compared to both spleen iNKT and PP CD4 cells. Red indicates higher expression and blue indicates lower expression.Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies, analyzed by flow cytometry and gated on CD1d (PBS57) tetramer+CD3+ cells.Quantification of data from D, n=11 mice per group. Mann-Whitney test. Error bars are SEM.


f1 ( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 μM ecdysone for 24 h . ( a ) The cell viability was assayed by trypan blue staining . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) . ( b ) Cell count and viability assay using Muse ( Millipore , USA ) . Data are mean from three independent experiments , with error bars representing s . d . * P0 . 05 ( Student ' s t - test ) . ( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 µM ecdysone for 24 h . ( c ) Caspase activity was measured from total cell lysate on DEVD - AMC , represented as fold increase relative to control RNAi . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) . ( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 μM ecdysone for 24 h . ( d ) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49 . Relative dUTX expression levels following knockdown using the two independent dsRNA are shown ( n = 3 ) with error bars representing s . d . * P0 . 05 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (a) The cell viability was assayed by trypan blue staining. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test). (b) Cell count and viability assay using Muse (Millipore, USA). Data are mean from three independent experiments, with error bars representing s.d. *P0.05 (Student's t-test).(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 µM ecdysone for 24 h. (c) Caspase activity was measured from total cell lysate on DEVD-AMC, represented as fold increase relative to control RNAi. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (d) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49. Relative dUTX expression levels following knockdown using the two independent dsRNA are shown (n=3) with error bars representing s.d. *P0.05 (Student's t-test).


f2 ( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( a ) GST and GST - dUTX - NR containing the nuclear hormone domain were used in pull - down experiments with 35S - labelled EcR and Usp in the presence of ethanol control ( − ) or 10 μM ecdysone ( + ) . The GST input proteins are shown in Coomassie Brilliant blue - stained gel , GST is labelled with arrow head and dUTX - NR with an asterisk . The EcR and Usp inputs are shown labelled with arrows . ( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( b ) Immunoprecipitation ( IP ) of EcR and dUTX in SL2 cells . Cells were transfected with HA - dUTX and the cell lysates were immunoprecipitated using anti - HA or a control IgG isotype antibody . The immunoprecipitates were analysed by western blotting using the anti - EcR and anti - HA antibodies . ( c ) Colocalization of dUTX and EcR on salivary gland polytene chromosomes . Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG - dUTX with dUTX ( green ) and EcR ( red ) , with Hoechst - stained DNA ( blue ) . Arrows indicate examples of colocalized bands . No signal was detected when immunostaining with secondary antibody only . Scale bar represent 5 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (a) GST and GST-dUTX-NR containing the nuclear hormone domain were used in pull-down experiments with 35S-labelled EcR and Usp in the presence of ethanol control (−) or 10 μM ecdysone (+). The GST input proteins are shown in Coomassie Brilliant blue-stained gel, GST is labelled with arrow head and dUTX-NR with an asterisk. The EcR and Usp inputs are shown labelled with arrows.(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (b) Immunoprecipitation (IP) of EcR and dUTX in SL2 cells. Cells were transfected with HA-dUTX and the cell lysates were immunoprecipitated using anti-HA or a control IgG isotype antibody. The immunoprecipitates were analysed by western blotting using the anti-EcR and anti-HA antibodies.(c) Colocalization of dUTX and EcR on salivary gland polytene chromosomes. Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG-dUTX with dUTX (green) and EcR (red), with Hoechst-stained DNA (blue). Arrows indicate examples of colocalized bands. No signal was detected when immunostaining with secondary antibody only. Scale bar represent 5 μm.


f3 ( a ) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF . Ovals indicate the position of salivary glands and fragments . Scale bar represent 50 μm . Quantification of the salivary gland phenotypic data was at 24 h RPF . ( b , c ) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF . ( b ) Cleaved caspase - 3 antibody ( green ) and Lamin antibody ( red ) staining are shown in control and dUTX1 salivary glands at 14 h RPF . Scale bar represents 50 μm . ( c ) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD - AMC as a substrate . Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts . The error bars represent s . e . m . * P - value0 . 05 ( Student ' s t - test ) . ( d ) Autophagy puncta examined using mCherry - Atg8a ( red ) and nuclei stained with Hoechst ( blue ) . Scale bar represents 100 μm . The quantitation of the mCherry - Atg8a puncta as the mean fluorescent pixel intensity per cell ( control n = 242 , dUTX1 n = 224 ) , with error bars representing s . e . m . * P - value0 . 05 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data was at 24 h RPF.(b,c) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF. (b) Cleaved caspase-3 antibody (green) and Lamin antibody (red) staining are shown in control and dUTX1 salivary glands at 14 h RPF. Scale bar represents 50 μm. (c) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD-AMC as a substrate. Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts. The error bars represent s.e.m. *P-value0.05 (Student's t-test).(d) Autophagy puncta examined using mCherry-Atg8a (red) and nuclei stained with Hoechst (blue). Scale bar represents 100 μm. The quantitation of the mCherry-Atg8a puncta as the mean fluorescent pixel intensity per cell (control n=242, dUTX1 n=224), with error bars representing s.e.m. *P-value0.05 (Student's t-test).


f4 ( a ) Levels of dark , dronc , drice and rpr transcripts were measured from third instar larvae ( L3 ) , 6 h and 14 h RPF salivary glands from control ( blue ) and dUTX1 ( red ) by qPCR and expressed relative to the internal control gene rp49 . ( b ) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control ( blue ) and dUTX1 ( red ) were analysed by qPCR at L3 , 6 h and 14 h RPF . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4(a) Levels of dark, dronc, drice and rpr transcripts were measured from third instar larvae (L3), 6 h and 14 h RPF salivary glands from control (blue) and dUTX1 (red) by qPCR and expressed relative to the internal control gene rp49.(b) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control (blue) and dUTX1 (red) were analysed by qPCR at L3, 6 h and 14 h RPF. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


f5 ( a , b ) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation ( ChIP ) . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess the binding to the promoters of dark , dronc , drice and rpr ( a ) ( a , b ) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation ( ChIP ) . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess the binding to the promoters of Atg1 , Atg2 , Atg3 , Atg4 , Atg5 , Atg6 , Atg7 , Atg8a , Atg9 , Atg12 and Atg18 ( b ) , expressed relative to the internal control gene Gapdh . Results are shown as the fold enrichment of % input compared with IgG control . ( c ) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP . SL2 cells knocked down for dUTX were chromatin - immunoprecipitated with anti - H3K27me3 and anti - GFP ( IgG control ) antibodies following ecdysone treatment . Results are shown as the fold enrichment of % input compared with RNAi control . ( d ) The occupancy of EcR on the promoter regions of dark , dronc and Atg1 by ChIP expressed relative to the internal control gene rp49 . Chromatin was immunoprecipitated with anti - EcR - B1 and anti - GFP ( IgG control ) antibodies from SL2 cell following ecdysone treatment . Results are shown as the fold enrichment of % input compared with IgG control . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of dark, dronc, drice and rpr (a)(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of Atg1, Atg2, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg9, Atg12 and Atg18 (b), expressed relative to the internal control gene Gapdh. Results are shown as the fold enrichment of % input compared with IgG control.(c) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP. SL2 cells knocked down for dUTX were chromatin-immunoprecipitated with anti-H3K27me3 and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control.(d) The occupancy of EcR on the promoter regions of dark, dronc and Atg1 by ChIP expressed relative to the internal control gene rp49. Chromatin was immunoprecipitated with anti-EcR-B1 and anti-GFP (IgG control) antibodies from SL2 cell following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).