f7WT and NRBF2 KO MEFs were treated with DMSO , 1 μM thapsigargin ( TP ) or 1 μM tunicamycin ( TN ) for 24 or 48 h . ( a ) Then cells were either imaged under phase - contrast microscope or ( b ) stained with propidium iodide ( PI ) and subjected to cell death rate analysis by flow cytometry . The representative images of cell morphology after treatments and quantitative analysis of PI - positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 5 ) . Scale bars , 100 μm ; Con , control . ( c ) Re - introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress . NRBF2 - CFP stably expressing cells were established in the NRBF2 KO background . Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry . The cell death rate in CFP - positive ( + ) population ( NRBF2 - CFP expression ) and negative ( − ) population ( no NRBF2 - CFP expression ) were recorded and analysed , respectively . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 3 ) . ( d ) ER stress inducers trigger caspase - 8 and caspase - 3 activation in NRBF2 KO MEFs . MEFs were treated with DMSO , 1 μM TP or 1 μM TN for 24 h , and then immunoprobed with indicated antibodies . Marked accumulation of cleaved caspase - 8 ( C - Cas 8 ; p43 and p18 units ) and cleaved caspase - 3 ( C - Cas 3 ) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN . This is a representative image from three independent experiments . ( e ) C - Cas 8 preferentially localizes at p62 aggregation foci . MEFs were treated with DMSO or 1 μM TP for 24 h , and then co - stained with an anti - p62 antibody and an anti - cleaved caspase - 8 antibody . The C - Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci . Scale bars , 20 μm . This is a representative image from three independent experiments .

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

f7WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin(TP) or 1 μM tunicamycin (TN) for 24 or 48 h. (a) Then cells were either imaged under phase-contrast microscope or (b) stained with propidium iodide (PI) and subjected to cell death rate analysis by flow cytometry. The representative images of cell morphology after treatments and quantitative analysis of PI-positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=5). Scale bars, 100 μm; Con, control.(c) Re-introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress. NRBF2-CFP stably expressing cells were established in the NRBF2 KO background. Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry. The cell death rate in CFP-positive (+) population (NRBF2-CFP expression) and negative (−) population (no NRBF2-CFP expression) were recorded and analysed, respectively. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=3).(d) ER stress inducers trigger caspase-8 and caspase-3 activation in NRBF2 KO MEFs. MEFs were treated with DMSO, 1 μM TP or 1 μM TN for 24 h, and then immunoprobed with indicated antibodies. Marked accumulation of cleaved caspase-8 (C-Cas 8; p43 and p18 units) and cleaved caspase-3 (C-Cas 3) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN. This is a representative image from three independent experiments.(e) C-Cas 8 preferentially localizes at p62 aggregation foci. MEFs were treated with DMSO or 1 μM TP for 24 h, and then co-stained with an anti-p62 antibody and an anti-cleaved caspase-8 antibody. The C-Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci. Scale bars, 20 μm. This is a representative image from three independent experiments.


f8 ( a ) NRBF2 is critical for the assembly of Vps34 - Vps15 and Atg14L - Beclin 1 complex . Brain lysate from WT and NRBF2 KO mice were subjected to co - IP by Vps34 antibody and Atg14L antibody . The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies . ( b , c ) Quantification results shows that Vps34 - Vps15 and Atg14L - Beclin 1 complex interactions as well as Atg14L - linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 4 mice in each group ; NS , not significant ) . ( d ) Overexpression of NRBF2 enhances overall Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP . Total Vps34 was pulled down by an anti - myc antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of IPed Vps34kinase activity , Vps15 protein normalized with IPed Vps34 shows that NRBF2 - CFP , but not CFP , dMIT - CFP or dCCD - CFP significantly enhance Vps34kinase activity and Vps34 - Vps15 interaction . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures . IPed Vps15 / IPed Vps34 , one sample t - test versus hypothetical mean 1 , Bonferroni correction , n = 3 ; IPed Vps34kinase activity / IPed Vps34 , multiple t - test followed by Bonferroni correction , n = 3 ) .

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 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

f8(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies. (b,c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=4 mice in each group; NS, not significant).(d) Overexpression of NRBF2 enhances overall Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34kinase activity and Vps34-Vps15 interaction. (Data are shown as mean±s.e.m., P values are indicated on the figures. IPed Vps15/IPed Vps34, one sample t-test versus hypothetical mean 1, Bonferroni correction, n=3; IPed Vps34kinase activity/IPed Vps34, multiple t-test followed by Bonferroni correction, n=3).


Figure 1b - d ) Comparative characterization of CHO cell bulk transfection cultures , selected for stable expression of the UnaG - or EGFP - encoding reporter constructs . b ) Flow cytometry revealed efficient induction of green fluorescence , after treatment of both UnaG and EGFP - expressing CHO bulk cultures with CoCl2 , while growth under hypoxia ( 1 % oxygen ) for 12 hours , selectively induced green fluorescence only in dUnaG expressing cells . Under normoxia ( 21 % oxygen ) only background fluorescence was observed . c ) Assessment of the mean fluorescence intensity ( MFI ) to determine the activity of the 5xHRE - CMV promoter after 12 and 24hrs . Fluorescence intensity increased between 12 and 24hrs . d ) Representative images of dUnaG - or dEGFP - expressing CHO cells grown either under normoxia ( 21 % oxygen ) , CoCl2 - treatment or hypoxia ( 1 % oxygen ) for 12 hours . Scale bars = 50µme ) Fluorescence intensity in dUnaG - or dEGFP - expressing CHO cell bulks grown under increasingly hypoxic conditions of 10 % , 5 % and 1 % oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5 % . f ) Maximal fluorescence of the UnaG - based hypoxia reporter in CHO cells grown at 1 % oxygen for 24 hrs hours was only observed at serum concentrations of 5 % FCS or higher , reflecting the dependence of UnaG on its serum cofactor bilirubin .

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 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 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 1b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs.d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µme) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%.f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.


Figure 2b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP . Expression under hypoxia was induced by incubation in 1 % oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia . Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly - destabilized UnaG - variants , while the doubly destabilized doUnaG displayed the most rapid switching behavior , but the least brightness . To reduce phototoxicity , illumination intensity was kept minimal . Data points represent one viewfield of 425 µm2 ± SEM . c ) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG . Open bars represent control cultures grown under normoxia . Solid bars indicate culture for 4 , 8 , 12 , 16 , 20 or 24 hrs under hypoxia ( 1 % oxygen ) . To assess deactivation , cultures previously kept under hypoxia ( 1 % oxygen ) for 24hrs were shifted to normoxia ( 21 % oxygen ) for the indicated times . Error bars represent standard errors . d ) Representative maximum intensity projections ( MIPs ) from the life cell cultures depicted in b . Cells were grown for the indicated times at hypoxic ( 1 % oxygen ) conditions and subsequently switched for the indicated times to normoxia ( 21 % oxygen ) ( red arrows in b ) to illustrate the dynamics of the different UnaG - based hypoxia sensors . Increasing destabilization is associated with reduced brightness but , also reduced background and improved switching behavior . Scale bars = 50 µm .

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Figure 2b) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM.c) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors.d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.


Figure 3a ) UnaG - based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model . 500 Gli36 glioblastoma cells , constitutively expressing mCherry and stably transfected with the HRE - dUnaG sensor construct , were stereotactically transplanted into the cortex of a SCIDmouse . Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation . Tumor cells are distinguished from the surrounding cortex by mCherry expression . Blood vessels were contrasted by immunostaining against PECAM - 1 . Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density ( outlined by white line in the composite panel ( bottom right ) . b ) Representative area from the tumor shown in a ) , which is located outside the viewfield in a ) positioned more closely to the tumor border . Also at the tumor border UnaG - expressing cells are preferentially observed at a distance to PECAM - 1 + vessels . c ) Areas of dUnaG expression correlate well with HIF - 1α stabilization . Immunostaining for HIF - 1α ( cyan ) revealed the predominantly nuclear localization of stabilized HIF - 1α in cells that were also dUnaG - positive ( green ) . d ) Analysis of dUnaG expressing cells in c ) for mCherry fluorescence and HIF - 1α stabilization . dUnaG - positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF - 1α The threshold was set in either case to channel 70 of 256 intensity channels . Using this classification > 60 % of UnaG positive cells displayed only background level mCherry fluorescence . On the other hand , > 98 % of the UnaG expressing cells also expressed HIF - 1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo . e ) The dUnaG - based senor was efficiently applied in intravital microscopy . 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG - sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy . We noted two distinct areas of intense UnaG fluorescence ( red lines ) . Shown is a MIP of a 60 µm z stack . The vasculature was contrasted by intravenous injection of non - functionalized Qdots . f ) Depth color coding of the MIP shown in e ) revealed the spatial relation between the vasculature , ( contrasted by Qdots ) , areas of increased dUnaG expression ( bordered by green lines ) and tumor cells expressing residual levels of mCherry . The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner ( red asterisk ) and the smaller vessels with a more axial orientation ( white arrows ) . Scale bars =

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 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 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

Figure 3a) UnaG-based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model. 500 Gli36 glioblastoma cells, constitutively expressing mCherry and stably transfected with the HRE-dUnaG sensor construct, were stereotactically transplanted into the cortex of a SCIDmouse. Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation. Tumor cells are distinguished from the surrounding cortex by mCherry expression. Blood vessels were contrasted by immunostaining against PECAM-1. Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density (outlined by white line in the composite panel (bottom right).b) Representative area from the tumor shown in a), which is located outside the viewfield in a) positioned more closely to the tumor border. Also at the tumor border UnaG-expressing cells are preferentially observed at a distance to PECAM-1+ vessels.c) Areas of dUnaG expression correlate well with HIF-1α stabilization. Immunostaining for HIF-1α (cyan) revealed the predominantly nuclear localization of stabilized HIF-1α in cells that were also dUnaG-positive (green).d) Analysis of dUnaG expressing cells in c) for mCherry fluorescence and HIF-1α stabilization. dUnaG-positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF-1α The threshold was set in either case to channel 70 of 256 intensity channels. Using this classification >60% of UnaG positive cells displayed only background level mCherry fluorescence. On the other hand, >98% of the UnaG expressing cells also expressed HIF-1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo.e) The dUnaG-based senor was efficiently applied in intravital microscopy. 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG-sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy. We noted two distinct areas of intense UnaG fluorescence (red lines). Shown is a MIP of a 60 µm z stack. The vasculature was contrasted by intravenous injection of non-functionalized Qdots.f) Depth color coding of the MIP shown in e) revealed the spatial relation between the vasculature, (contrasted by Qdots), areas of increased dUnaG expression (bordered by green lines) and tumor cells expressing residual levels of mCherry. The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner (red asterisk) and the smaller vessels with a more axial orientation (white arrows). Scale bars = 100µm


Figure 4b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) in CHO cells stably transfected with the dUnOHR sensor . Hypoxia was induced by incubation in 1 % oxygen for 18 hrs , then the culture was switched to normoxia for 24 hrs , followed by another 14 hrs hypoxia and finally normoxia again . As expected UnaG fluorescence is efficiently induced under hypoxia , while mOrange fluorescence only appears after the switch to normoxia only . The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE - mCMV promoter activity . This behavior is repeated in subsequent hypoxia / normoxia cycles . The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period . Plotted is the average of the mean ± SEM . c ) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b . ) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange . Scale bars = 100 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 4b) Microscopic assessment of the averaged fluorescence intensity (AFI) in CHO cells stably transfected with the dUnOHR sensor. Hypoxia was induced by incubation in 1% oxygen for 18 hrs, then the culture was switched to normoxia for 24 hrs, followed by another 14 hrs hypoxia and finally normoxia again. As expected UnaG fluorescence is efficiently induced under hypoxia, while mOrange fluorescence only appears after the switch to normoxia only. The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE-mCMV promoter activity. This behavior is repeated in subsequent hypoxia / normoxia cycles. The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period. Plotted is the average of the mean ± SEM.c) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b.) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange. Scale bars = 100 µm.


Figure 5a ) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct . The perimeter of the developing tumor ( white line in the merged picture , lower right panel ) was delineated by Hoechst 33342 staining . As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density . Vascular endothelium was identified by PECAM - 1 staining . 30 µm cryosection . Scale bar = 500µm . b ) Details of the tumor shown in a ) . Sparse , distinct cells are characterized by intense green and orange fluorescence ( shown as orange ) and are located preferentially to the edge of the hypoxic phalanx ( white arrows ) . A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence ( asterisks ) , indicative of previously hypoxic environment and subsequent reoxygenation . Scale bar = 50 µm . c ) Recently reoxygenated cells ( arrows ) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber ( double arrows ) . Scale bar = 100 µm . d ) Previously hypoxic cells , frequently undergo apoptosis after reoxygenation . We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas . Scale bar = 50µm . e ) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis . Scale bar = 50µm .

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 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 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 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

Figure 5a) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct. The perimeter of the developing tumor (white line in the merged picture, lower right panel) was delineated by Hoechst 33342 staining. As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density. Vascular endothelium was identified by PECAM-1 staining. 30 µm cryosection. Scale bar = 500µm.b) Details of the tumor shown in a). Sparse, distinct cells are characterized by intense green and orange fluorescence (shown as orange) and are located preferentially to the edge of the hypoxic phalanx (white arrows). A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence (asterisks), indicative of previously hypoxic environment and subsequent reoxygenation. Scale bar = 50 µm.c) Recently reoxygenated cells (arrows) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber (double arrows). Scale bar = 100 µm.d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm.e) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis. Scale bar = 50µm.


Figure 1Ookinete formation in vitro . After 12 h of culture , the ookinetes were Giemsa - stained and analyzed for ookinete morphology . The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes . Oocyst counts in the mosquitoes at day 7 post blood - feeding . x / y on the top is the number of mosquitoes containing oocyst / the number of mosquitoes dissected ; the percentage number is the mosquito infection prevalence .

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Figure 1Ookinete formation in vitro. After 12 h of culture, the ookinetes were Giemsa-stained and analyzed for ookinete morphology. The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes.Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.


Figure 2Co - staining of P28 and GAP45 ( IMC protein ) in female gametes ( P28 + only ) and zygotes ( P28 + / GAP45 + ) of the 17XNL , Δap2 - o3 and Δcdpk4 strains . Percentages of female gametes and zygotes were indicated respectively . n is the number of cells counted . Scale bars = 5 μm . Note : the signal of P28 and GAP45 is reduced similarly in all Δap2 - o3 female gametes and zygotes compared to that of 17XNL . Day 7 midgut oocyst counts from mosquitoes infected with parasites , including 17XNL , Δap2 - o3 , Δmap2 , or Δnek4 strain alone as well as mixtures of Δmap2 / Δnek4 , Δap2 - o3 / Δmap2 , and Δap2 - o3 / Δnek4 . Δnek4 and Δmap2 are female and male gamete - defect parasites , respectively . n is the number of mosquitoes dissected , Mann - Whitney test applied , two experiments repeated .

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Figure 2Co-staining of P28 and GAP45 (IMC protein) in female gametes (P28+ only) and zygotes (P28+/GAP45+) of the 17XNL, Δap2-o3 and Δcdpk4 strains. Percentages of female gametes and zygotes were indicated respectively. n is the number of cells counted. Scale bars = 5 μm. Note: the signal of P28 and GAP45 is reduced similarly in all Δap2-o3 female gametes and zygotes compared to that of 17XNL.Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, Δap2-o3, Δmap2, or Δnek4 strain alone as well as mixtures of Δmap2/Δnek4, Δap2-o3/Δmap2, and Δap2-o3/Δnek4. Δnek4 and Δmap2 are female and male gamete-defect parasites, respectively. n is the number of mosquitoes dissected, Mann-Whitney test applied, two experiments repeated.


Figure 3Transcript expression heatmap by RNA - seq of 26 genes involving DNA replication and DNA repair between DFsc7 ( WT ) and DFsc7 ; Δap2 - o3 ( KO ) strains .

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Figure 3Transcript expression heatmap by RNA-seq of 26 genes involving DNA replication and DNA repair between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains.


Figure 4Representative fluorescence microscopy images of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 ( ccp2 : : mCherry ; dpod2 : : gfp ) and DTS1 ; Δap2 - o3 strains . mCherry is specifically expressed in female gametocytes . Flow cytometry detection of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 and DTS1 ; Δap2 - o3 strains . The female and male gametocyte populations are circled by solid and dashed line respectively . E , F Similar analysis ( as in C , D ) in DTS2 ( ccp2 : : mCherry ; dpod1 : : gfp ) and DTS2 ; Δap2 - o3 strains . G , H Similar analysis ( as in C , D ) in DTS3 ( ccp2 : : mCherry ; rpa1 : : gfp ) and DTS3 ; Δap2 - o3 strains . I , J Similar analysis ( as in C , D ) in DTS4 ( ccp2 : : mCherry ; mcm7 : : gfp ) and DTS4 ; Δap2 - o3 strains .

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Figure 4Representative fluorescence microscopy images of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 (ccp2::mCherry;dpod2::gfp) and DTS1;Δap2-o3 strains. mCherry is specifically expressed in female gametocytes. Flow cytometry detection of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 and DTS1;Δap2-o3 strains. The female and male gametocyte populations are circled by solid and dashed line respectively. E,F Similar analysis (as in C, D) in DTS2 (ccp2::mCherry;dpod1::gfp) and DTS2;Δap2-o3 strains. G,H Similar analysis (as in C, D) in DTS3 (ccp2::mCherry;rpa1::gfp) and DTS3;Δap2-o3 strains. I,J Similar analysis (as in C, D) in DTS4 (ccp2::mCherry;mcm7::gfp) and DTS4;Δap2-o3 strains.


Figure 5EMSA using the recombinant GST - fused AP2 domain of AP2 - O3 and a synthesized DNA probe containing three repeats of predicted motif . GST was used as a negative control . An arrowhead indicates the shifted band . ChIP - qPCR validation of the binding between AP2 - O3 and the upstream region of four male genes The regions detected via ChIP - qPCR are indicated by pink line ( in the peak ) and purple line ( out of the peak ) . mean ± SEM from three independent experiments .

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Figure 5EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band.ChIP-qPCR validation of the binding between AP2-O3 and the upstream region of four male genes The regions detected via ChIP-qPCR are indicated by pink line (in the peak) and purple line (out of the peak). mean±SEM from three independent experiments.


Figure 6qRT - PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID . Western blot of GAP45 protein expression in non - activated ( NAG ) and activated gametocyte ( AG ) of 17XNL and Δap2 - o3 strains . BiP as loading control .

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Figure 6qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID.Western blot of GAP45 protein expression in non-activated (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control.


Figure 7IFA of AP2 - O3 protein expression in activated gametocytes of Pccp2 strains . Oocyst counts in the mosquitoes at day 7 post blood - feeding . x / y on the top is the number of mosquitoes containing oocyst / the number of mosquitoes dissected ; the percentage number is the mosquito infection prevalence .

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Figure 7IFA of AP2-O3 protein expression in activated gametocytes of Pccp2 strains.Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.


Figure 152 - day - old ( A ) plants of the genotypes indicated grown in LDs . Scale bars : 5 cm . B - C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In ( B - C data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) . D - E qPCR results showing mRNA expression of CIB1 , FT in the genotypes indicated . The numbers indicate independent transgenic lines . The transcript levels of CIB1 or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed . F . 25 - day - old plants ( F ) of the genotypes indicated grown in LDs . Scale bars : 5 cm . , G - H . The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In G - H ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) . , I - J . qPCR results showing mRNA expression of CO , FT in the genotypes indicated . The numbers indicate independent transgenic lines . The transcript levels of CO or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed .

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Figure 152-day-old (A) plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B-C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B-C data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).D-E qPCR results showing mRNA expression of CIB1, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CIB1 or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.F. 25-day-old plants (F) of the genotypes indicated grown in LDs. Scale bars: 5 cm. , G-H. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In G-H), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test)., I-J. qPCR results showing mRNA expression of CO, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CO or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


Figure 2Immunoblots showing CIB1 ( A ) protein levels in WT and co - 1 backgrounds ( A ) over the course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - actin antibody ( ACT ) . B . Immunoblots showing CO ( B ) protein levels in WT and cibh backgrounds ( B ) over the course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - actin antibody ( ACT ) . The relative levels of CIB1 in WT and co - 1 backgrounds over the course of 20 hours under LDs were calculated Data information : Error bars represent the standard deviation of three biological replicates of the Western blots . D . The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information : Error bars represent the standard deviation of three biological replicates of the Western blots .

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Figure 2Immunoblots showing CIB1 (A) protein levels in WT and co-1 backgrounds (A) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).B. Immunoblots showing CO (B) protein levels in WT and cibh backgrounds (B) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).The relative levels of CIB1 in WT and co-1 backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.D. The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.


Figure 3A . Fluorescent microscopy images showing that CIB1 co - localizes with CO in nuclear speckles . Scale bars : 2 μm . B . BiFC assays to show in vivo protein interactions . Epidermal cells of N . benthamiana leaves were co - transformated with cCFP - CO and nYFP - CIB1 , with cCFP - CO and nYFP , or with cCFP and nYFP - CIB1 . BF , bright field . Merge , overlay of the YFP and bright field images . Scale bars : 50 μm . Data information : For each experiment , two or more biological replicates were performed . C . Split - LUC assay showing that CIB1 interacts with CO . Leaf epidermal cells of N . benthamiana were co - transformed with CO - nLUC and cLUC - CIB1 or cLUC or nLUC with cLUC - CIB or cLUC . Data information : For each experiment , two or more biological replicates were performed . D . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 , 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag seedlings grown in LDs . Data information : For each experiment , two or more biological replicates were performed . F . In vitro pull - down assays showing the interaction between CIBC and CO , or CIBC and COΔN and the lack of interaction between CIB1C and COΔC . His - TF tagged CO , COΔN , COΔC or His - TF tagged UVR8 were mixed with GST - CIB1C purified from E . coli , and His antibody was used for the in vitro pull - down assay . The products were analyzed by immunoblots probed with the anti - CIB1C antibody or the anti - His antibody ( UVR8 , CO , COΔC , COΔN ) . Data information : For each experiment , two or more biological replicates were performed .

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Figure 3A. Fluorescent microscopy images showing that CIB1 co-localizes with CO in nuclear speckles. Scale bars: 2 μm.B. BiFC assays to show in vivo protein interactions. Epidermal cells of N. benthamiana leaves were co-transformated with cCFP-CO and nYFP-CIB1, with cCFP-CO and nYFP, or with cCFP and nYFP-CIB1. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bars: 50 μm. Data information: For each experiment, two or more biological replicates were performed.C. Split-LUC assay showing that CIB1 interacts with CO. Leaf epidermal cells of N. benthamiana were co-transformed with CO-nLUC and cLUC-CIB1 or cLUC or nLUC with cLUC-CIB or cLUC. Data information: For each experiment, two or more biological replicates were performed.D. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1, 35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag seedlings grown in LDs. Data information: For each experiment, two or more biological replicates were performed.F. In vitro pull-down assays showing the interaction between CIBC and CO, or CIBC and COΔN and the lack of interaction between CIB1C and COΔC. His-TF tagged CO, COΔN, COΔC or His-TF tagged UVR8 were mixed with GST-CIB1C purified from E. coli, and His antibody was used for the in vitro pull-down assay. The products were analyzed by immunoblots probed with the anti-CIB1C antibody or the anti-His antibody (UVR8, CO, COΔC, COΔN). Data information: For each experiment, two or more biological replicates were performed.


Figure 4A . 28 - day - old plants of the genotypes indicated grown in LDs . Scale bars : 5 cm . B , C . The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In ( B , C ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) . D . Immunoblots showing Myc - CIB1 and CO - Flag protein levels in the genotypes indicated . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - Flag antibody . E . qPCR results showing mRNA expression of FT in the genotypes indicated . The transcript levels in Col are set to 1 . 0 . Samples were collected from 7 - day - old seedlings of the genotypes indicated at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed . G . Box plots ( the minimum , first quartile , median , third quartile , and maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in plants expressing different reportors and effectors . The relative LUC activities normalized to the REN activity are shown . The LUC / REN in plants expressing ProFT alone is set to 1 . 0 . Data information : data are presented as mean ± SD , n = 4 . The letters " a " to " g " indicate statistically significant differences determined by Tukey ' s HSD test ( P ≤ 0 . 05 ) . Two biological replicates were performed .

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Figure 4A. 28-day-old plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B, C. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B, C), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).D. Immunoblots showing Myc-CIB1 and CO-Flag protein levels in the genotypes indicated. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-Flag antibody.E. qPCR results showing mRNA expression of FT in the genotypes indicated. The transcript levels in Col are set to 1.0. Samples were collected from 7-day-old seedlings of the genotypes indicated at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.G. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The relative LUC activities normalized to the REN activity are shown. The LUC/REN in plants expressing ProFT alone is set to 1.0. Data information: data are presented as mean ± SD, n=4. The letters "a" to "g" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.


Figure 5A . Fluorescent microscopy images showing that CRY2 , CIB1 and CO co - localized to the nucleus . Scale bars : 2 μB . Co - IP assays showing that CRY2 , CIB1 and CO form a protein complex . Samples were prepared from 10 - day - old WT or 35S : Myc - CIB1 / 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) , and samples were collected at ZT16 . C . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) . Samples were collected at the times indicated over the course of one day . E . Representative results of the ChIP - qPCR assays . Chromatin fragments ( ~ 500 bp ) were prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag seedlings , immunoprecipitated by the anti - CRY2 , anti - Flag ( CO - Flag ) , anti - Myc ( Myc - CIB1 ) antibody or anti - preimmune IgG , and the precipitated DNA was analyzed by qPCR using the primer pairs indicated . The IP / input ratio of anti - preimmune is set to 1 . 0 . Data information : the relative IP / input ratios are shown with their standard deviation ( n = 3 ) . Two biological replicates were performed . F . Box plots ( the minimum , first quartile , median , third quartile , and maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in plants expressing different reportors and effectors . The LUC / REN in plants expressing ProFT alone is set to 1 . 0 . Immunoblots were done to show GFP - CRY2W374A , Myc - CIB1 and Myc - CO protein levels in the transient Dual - LUC assay . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , and with the anti - GFP antibody . Data information : the relative LUC activities normalized to the REN activity are shown ( LUC / REN ) with their standard deviation ( n = 6 ) . The letters " a " to " f " indicate statistically significant differences determined by Tukey ' s HSD test ( P ≤ 0 . 05 ) . Two biological replicates were performed .

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 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 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 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

Figure 5A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μB. Co-IP assays showing that CRY2, CIB1 and CO form a protein complex. Samples were prepared from 10-day-old WT or 35S:Myc-CIB1/35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D), and samples were collected at ZT16.C. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D). Samples were collected at the times indicated over the course of one day.E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0. Data information: the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed.F. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The LUC/REN in plants expressing ProFT alone is set to 1.0. Immunoblots were done to show GFP-CRY2W374A, Myc-CIB1 and Myc-CO protein levels in the transient Dual-LUC assay. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, and with the anti-GFP antibody. Data information: the relative LUC activities normalized to the REN activity are shown (LUC/REN) with their standard deviation (n =6). The letters "a" to "f" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.


Figure 1Confocal z - projections of the indicated genotypes and age , labeled with membrane bound GFP ( mCD8 - GFP ; CD8 ) driven by the γ - specific Gal4 driver GMR71G10 - Gal4 ( γ - Gal4 ) . While γ - axons of control flies project through the entire lobe ( C is the RNAi control ; n = 12 / 12 , E ; is the tsCRISPR control ; n = 14 / 14 ) , knockdown of dpr12 by RNAi ( D ; n = 12 / 12 ) or knockout by tsCRISPR ( F ; n = 14 / 14 ) resulted in short axons . At L3 , γ - axons in dpr12 ∆ 50 - 81 homozygotus mutant animals ( K ; n = 20 / 20 ) resemble WT γ - axons ( G ; n = 20 / 20 ) . At 48hr APF , γ - axons normally re - extend to form the adult lobe ( H ; n = 12 / 12 ) . dpr12 ∆ 50 - 81 γ - axons ( L ; n = 14 / 14 ) fail to extend to the end of the lobe . This defect persists to adult ( I ; n = 11 / 11 vs . M ; n = 18 / 18 ) . Expressing a UAS - Dpr12 transgene within γ - KCs in dpr12 ∆ 50 - 81 homozygote mutant animals rescued the axon regrowth defect ( N ; n = 23 / 24 , J ; n = 14 / 14 ) . The adult γ - lobe and α / β lobes are outlined in ( C , D ) in yellow and orange , respectively , for clarity . Asterisks demarcate the distal part of the lobe . Green and white indicate mCD8 - GFP . Magenta represents FasII staining . Scale bar is 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Confocal z-projections of the indicated genotypes and age, labeled with membrane bound GFP (mCD8-GFP; CD8) driven by the γ-specific Gal4 driver GMR71G10-Gal4 (γ-Gal4). While γ-axons of control flies project through the entire lobe (C is the RNAi control; n=12/12, E; is the tsCRISPR control; n=14/14), knockdown of dpr12 by RNAi (D; n=12/12) or knockout by tsCRISPR (F; n=14/14) resulted in short axons. At L3, γ-axons in dpr12∆50-81 homozygotus mutant animals (K; n=20/20) resemble WT γ-axons (G; n=20/20). At 48hr APF, γ-axons normally re-extend to form the adult lobe (H; n=12/12). dpr12∆50-81 γ-axons (L; n=14/14) fail to extend to the end of the lobe. This defect persists to adult (I; n=11/11 vs. M; n=18/18). Expressing a UAS-Dpr12 transgene within γ-KCs in dpr12∆50-81 homozygote mutant animals rescued the axon regrowth defect (N; n=23/24, J; n=14/14). The adult γ-lobe and α/β lobes are outlined in (C, D) in yellow and orange, respectively, for clarity. Asterisks demarcate the distal part of the lobe. Green and white indicate mCD8-GFP. Magenta represents FasII staining. Scale bar is 20 µm.


Figure 2Confocal z - projections of MARCM neuroblast ( NB , A - F ) and single - cell ( SC , G - L ) clones labeled with membrane bound GFP ( mCD8 - GFP ; CD8 ) driven by the γ specific Gal4 driver GMR71G10 - Gal4 ( γ - Gal4 ) . At L3 , NB and SC clones expressing dpr12 RNAi are similar to equivalent WT clones ( A ; n = 20 / 20 , D ; n = 15 / 15 , G ; n = 15 / 15 and J ; n = 17 / 17 ) . At 48hr APF and adult stage , WT NB ( B ; n = 15 / 15 , C ; n = 10 / 10 ) and SC ( H ; n = 16 / 16 , I ; n = 13 / 13 ) clones extend their axons to form the full adult lobe . In contrast , clones expressing dpr12 RNAi ( E ; n = 14 / 14 , F ; n = 22 / 2 , K ; n = 18 / 24 , L ; n = 19 / 27 ) fail to extend their axons to the distal part of the medial lobe ( asterisks ) . I ' and L ' are traces of multiple single - cell clones depicting each cell in a different color .

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

Figure 2Confocal z-projections of MARCM neuroblast (NB, A-F) and single-cell (SC, G-L) clones labeled with membrane bound GFP (mCD8-GFP; CD8) driven by the γ specific Gal4 driver GMR71G10-Gal4 (γ-Gal4). At L3, NB and SC clones expressing dpr12 RNAi are similar to equivalent WT clones (A; n=20/20, D; n=15/15, G; n=15/15 and J; n=17/17). At 48hr APF and adult stage, WT NB (B; n=15/15, C; n=10/10) and SC (H; n=16/16, I; n=13/13) clones extend their axons to form the full adult lobe. In contrast, clones expressing dpr12 RNAi (E; n=14/14, F; n=22/2, K; n=18/24, L; n=19/27) fail to extend their axons to the distal part of the medial lobe (asterisks). I' and L' are traces of multiple single-cell clones depicting each cell in a different color.


Figure 3Confocal z - projections of brains expressing MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ; A - D ) and DIP - δGFSTF ( DIP - δ - GFP ; E - H ) fusion proteins at the indicated time points . See Figures EV 1 and 4 for more details on the fusion protein structure . ( A - D ) Dpr12 - GFP is localized to the distal part of the γ - lobe at L3 ( A ; n = 10 / 10 ) , 48hr APF ( C ; n = 12 / 12 ) and the adult stage ( D ; n = 20 / 20 ) , where it colocalizes with the γ4 > γ1γ2 MBON ( γ4 ; labeled by GMR18H09 - Gal4 driving the expression of CD4 - tdT ) . At 24hr APF ( B ; n = 10 / 10 ) , Dpr12 - GFP appears diffuse . ( E - H ) DIP - δ - GFP is localized to the distal part of the γ - lobe throughout development : L3 ( E ; n = 16 / 16 ) , 24hr APF ( F ; n = 10 / 10 ) , 48hr APF ( G ; n = 12 / 12 ) and adult ( H ; n = 24 / 24 ) . At the adult stage , DIP - δ - GFP is colocalized with the γ4 > γ1γ2 MBON ( γ4 ) .

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

Figure 3Confocal z-projections of brains expressing MiMIC mediated Dpr12GFSTF (Dpr12-GFP; A-D) and DIP-δGFSTF (DIP-δ-GFP; E-H) fusion proteins at the indicated time points. See Figures EV 1 and 4 for more details on the fusion protein structure. (A-D) Dpr12-GFP is localized to the distal part of the γ-lobe at L3 (A; n=10/10), 48hr APF (C; n=12/12) and the adult stage (D; n=20/20), where it colocalizes with the γ4>γ1γ2 MBON (γ4; labeled by GMR18H09-Gal4 driving the expression of CD4-tdT). At 24hr APF (B; n=10/10), Dpr12-GFP appears diffuse. (E-H) DIP-δ-GFP is localized to the distal part of the γ-lobe throughout development: L3 (E; n=16/16), 24hr APF (F; n=10/10), 48hr APF (G; n=12/12) and adult (H; n=24/24). At the adult stage, DIP-δ-GFP is colocalized with the γ4>γ1γ2 MBON (γ4).


Figure 4Confocal z - projections DIP - δ hetero - and homozygous brains in which γ - KCs were labeled by expressing membrane bound tandem tomato ( mtdT - HA ) driven by the γ specific QF2 driver R71G10 - QF2 ( γ - QF2 ) . Larval ( L3 ) γ - axons grow normally in DIP - δT2A - Gal4 heterozygotes ( A ; n = 16 / 16 ) and homozygotes ( E ; n = 24 / 24 ) . In contrast , at 48hr APF and adult , γ - axons within DIP - δT2A - Gal4 homozygotes do not enter the distal part of the lobe ( asterisks ; F ; n = 8 / 8 , G ; n = 20 / 20 ) , while they grow normally in heterozygotes ( B ; n = 12 / 12 , C ; n = 12 / 12 ) . Overexpression of a DIP - δ transgene driven by the Gal4 activity of DIP - δT2A - Gal4 ( see also Figure S4 ) does not affect normal growth ( D ; n = 10 / 10 ) and rescues mutant phenotypes ( H ; n = 20 / 20 ) . Note that while the R71G10 driver is consistently expressed in γ - KCs , it is also expressed in α / β - KCs in a stochastic manner . The adult γ - lobe and α / β lobes are outlined in C - D in yellow and orange , respectively , for clarity . Confocal z - projections of brains expressing DIP - δ - RNAi driven by the indicated Gal4 . γ - KCs are labeled by mtdT - HA driven by R71G10 - QF2 ( γ - QF2 ) . Expression of DIP - δ - RNAi in all glia ( Repo - Gal4 , J ; n = 28 / 28 ) or all KCs ( OK107 - Gal4 , K ; n = 12 / 12 ) did not affect γ - neuron regrowth . In contrast , expression of DIP - δ - RNAi in all postmitotic neurons ( C155 - Gal4 , L ; n = 9 / 12 ) or DIP - δ expressing neurons ( DIP - δT2A - Gal4 , M ; n = 21 / 22 ) induced a defect in γ4 / 5 innervation by γ - axons .

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 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

Figure 4Confocal z-projections DIP-δ hetero- and homozygous brains in which γ-KCs were labeled by expressing membrane bound tandem tomato (mtdT-HA) driven by the γ specific QF2 driver R71G10-QF2 (γ-QF2). Larval (L3) γ-axons grow normally in DIP-δT2A-Gal4 heterozygotes (A; n=16/16) and homozygotes (E; n=24/24). In contrast, at 48hr APF and adult, γ-axons within DIP-δT2A-Gal4 homozygotes do not enter the distal part of the lobe (asterisks; F; n=8/8, G; n=20/20), while they grow normally in heterozygotes (B; n=12/12, C; n=12/12). Overexpression of a DIP-δ transgene driven by the Gal4 activity of DIP-δT2A-Gal4 (see also Figure S4) does not affect normal growth (D; n=10/10) and rescues mutant phenotypes (H; n=20/20). Note that while the R71G10 driver is consistently expressed in γ-KCs, it is also expressed in α/β-KCs in a stochastic manner. The adult γ-lobe and α/β lobes are outlined in C-D in yellow and orange, respectively, for clarity.Confocal z-projections of brains expressing DIP-δ-RNAi driven by the indicated Gal4. γ-KCs are labeled by mtdT-HA driven by R71G10-QF2 (γ-QF2). Expression of DIP-δ-RNAi in all glia (Repo-Gal4, J; n=28/28) or all KCs (OK107-Gal4, K; n=12/12) did not affect γ-neuron regrowth. In contrast, expression of DIP-δ-RNAi in all postmitotic neurons (C155-Gal4, L; n=9/12) or DIP-δ expressing neurons (DIP-δT2A-Gal4, M; n=21/22) induced a defect in γ4/5 innervation by γ-axons.


Figure 5 ( A - D ) Confocal z - projections of brains expressing DIP - δGFSTF ( DIP - δ - GFP ) together with the indicated Gal4s and transgenes . Expressing diphtheria toxin ( DTi ) and the membrane bound RFP ( mCD8 - RFP ; CD8 ) driven by the γ4 > γ1γ2 MBON driver MB294B - Gal4 ( MBONγ4 > γ1γ2 - Gal4 ) did not affect DIP - δ - GFP expression ( A , n = 16 / 16 ; B , n = 14 / 14 ) . In contrast , similar expression of DTi and membrane bound Tomato ( CD4 - tdT ; CD4 ) in PAM - DANs ( using the GMR58E02 - Gal4 ; PAM - DAN - Gal4 ) abolished the normal DIP - δ - GFP expression in the γ4 / 5 zone ( compare D , n = 18 / 18 , to C , n = 16 / 16 ) and within the PAM - DAN cell bodies ( compare D ' to C ' ) . Magenta is CD8 - RFP ( A - B ) , CD4 - mtdT ( C - D ) . Green is GFP , grayscale depict individual channels as labeled . Scale bar is 20 µm . Yellow dashed line demarcates the γ - lobe based on FasII staining ( not shown ) . ( E - G ) Confocal z - projections of MARCM clones labeled by DIP - δT2A - Gal4 ( DIP - δ Gal4 ) driving the expression of membrane bound GFP ( mCD8 - GFP ; CD8 ) and heat shocked at 24hr after egg laying . Clones innervate the γ4 / 5 zones at 24hr APF ( E ; n = 8 ) , 48hr APF ( F ; n = 8 ) and adult ( G ; n = 16 ) . Clones become tyrosine hydroxylase ( TH ) positive only at 48hr APF onwards ( F , G ) . Magenta is FasII , green is mCD8 - GFP , cyan is TH antibody staining , grayscale single channels are shown as indicated . White dashed line demarcates the γ - lobe . Scale bar is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 5(A-D) Confocal z-projections of brains expressing DIP-δGFSTF (DIP-δ-GFP) together with the indicated Gal4s and transgenes. Expressing diphtheria toxin (DTi) and the membrane bound RFP (mCD8-RFP; CD8) driven by the γ4>γ1γ2 MBON driver MB294B-Gal4 (MBONγ4>γ1γ2-Gal4) did not affect DIP-δ-GFP expression (A, n=16/16; B, n=14/14). In contrast, similar expression of DTi and membrane bound Tomato (CD4-tdT; CD4) in PAM-DANs (using the GMR58E02-Gal4; PAM-DAN-Gal4) abolished the normal DIP-δ-GFP expression in the γ4/5 zone (compare D, n=18/18, to C, n=16/16) and within the PAM-DAN cell bodies (compare D' to C'). Magenta is CD8-RFP (A-B), CD4-mtdT (C-D). Green is GFP, grayscale depict individual channels as labeled. Scale bar is 20 µm. Yellow dashed line demarcates the γ-lobe based on FasII staining (not shown).(E-G) Confocal z-projections of MARCM clones labeled by DIP-δT2A-Gal4 (DIP-δ Gal4) driving the expression of membrane bound GFP (mCD8-GFP; CD8) and heat shocked at 24hr after egg laying. Clones innervate the γ4/5 zones at 24hr APF (E; n=8), 48hr APF (F; n=8) and adult (G; n=16). Clones become tyrosine hydroxylase (TH) positive only at 48hr APF onwards (F,G). Magenta is FasII, green is mCD8-GFP, cyan is TH antibody staining, grayscale single channels are shown as indicated. White dashed line demarcates the γ-lobe. Scale bar is 20 µm.


Figure 6 ( A - J ) Confocal z - projections of brains expressing MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ) and DIP - δGFSTF ( DIP - δ - GFP ) fusion proteins of the indicated genotypes and time points . ( A - D ) Dpr12 - GFP expression is diffuse in DIP - δT2A - Gal4 homozygotes mutant brains at L3 ( A ; n = 20 / 20 ) , 24hr APF ( B ; n = 14 / 14 ) , 48hr APF ( C ; n = 28 / 28 ) and adult ( D ; n = 26 / 26 ) . ( E - H ) DIP - δ - GFP expression in dpr12 ∆ 50 - 81 homozygotes mutant brains remains localized to the distal part of the γ - lobe at L3 ( E ; n = 16 / 16 ) , 24hr APF ( F ; n = 16 / 16 ) , and 48hr APF ( G ; n = 10 / 10 ) but cannot be identified in adult brains ( H ; n = 16 / 16 ) . ( I - J ) Dpr12 - GFP expression in WT animals ( I , n = 8 / 8 ) or in those ectopically expressing DIP - δ in PAM - DANs that innervate the γ3 zone ( J , n = 14 / 14 ) driven by MB441B - Gal4 ( PAM - DAN - γ3 - Gal4 ) . DIP - δ expression in PAM - DAN - γ3 resulted in Dpr12 - GFP localization within the γ3 zone ( arrow ) , in addition to its normal γ4 / γ5 localization .

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

Figure 6(A-J) Confocal z-projections of brains expressing MiMIC mediated Dpr12GFSTF (Dpr12-GFP) and DIP-δGFSTF (DIP-δ-GFP) fusion proteins of the indicated genotypes and time points. (A-D) Dpr12-GFP expression is diffuse in DIP-δT2A-Gal4 homozygotes mutant brains at L3 (A; n=20/20), 24hr APF (B; n=14/14), 48hr APF (C; n=28/28) and adult (D; n=26/26). (E-H) DIP-δ-GFP expression in dpr12∆50-81 homozygotes mutant brains remains localized to the distal part of the γ-lobe at L3 (E; n=16/16), 24hr APF (F; n=16/16), and 48hr APF (G; n=10/10) but cannot be identified in adult brains (H; n=16/16). (I-J) Dpr12-GFP expression in WT animals (I, n=8/8) or in those ectopically expressing DIP-δ in PAM-DANs that innervate the γ3 zone (J, n=14/14) driven by MB441B-Gal4 (PAM-DAN-γ3-Gal4). DIP-δ expression in PAM-DAN-γ3 resulted in Dpr12-GFP localization within the γ3 zone (arrow), in addition to its normal γ4/γ5 localization.


Figure 7Confocal z - projections of dpr12 ∆ 50 - 81 heterozygous ( A , n = 15 ; C , n = 10 ; E , n = 10 ) and homozygous brains ( B , n = 8 ; D , n = 18 ; F , n = 24 ) expressing mCD8 - GFP ( CD8 ) driven by ( E , F ) R18H09 - Gal4 ( MBONγ4 > γ1γ2 - Gal4 ) . The greyscale channels are sub - z - projections comprised of slices restricted to the γ - lobe region . White dashed line demarcates the γ - lobe . Bottom : Cartoons schematizing MB lobe structure and innervation by specific PAM - DANs or MBON . Single confocal slices of WT ( G , n = 5 / 5 ) and dpr12 ∆ 50 - 81 homozygous brains ( H , n = 5 / 5 ) stained with anti - Brp . Dashed lines demarcate neuropil boundaries , schematic shown below . Cre , crepine , a neuropil that surrounds the medial MB lobes ; AL , antenna lobe .

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 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

Figure 7Confocal z-projections of dpr12∆50-81 heterozygous (A, n=15; C, n=10; E, n=10) and homozygous brains (B, n=8; D, n=18; F, n=24) expressing mCD8-GFP (CD8) driven by (E, F) R18H09-Gal4 (MBONγ4>γ1γ2 -Gal4). The greyscale channels are sub-z-projections comprised of slices restricted to the γ-lobe region. White dashed line demarcates the γ-lobe. Bottom: Cartoons schematizing MB lobe structure and innervation by specific PAM-DANs or MBON.Single confocal slices of WT (G, n=5/5) and dpr12∆50-81 homozygous brains (H, n=5/5) stained with anti-Brp. Dashed lines demarcate neuropil boundaries, schematic shown below. Cre, crepine, a neuropil that surrounds the medial MB lobes; AL, antenna lobe.


Figure 8 ( C , D ) Confocal z - projections of adult DIP - δT2A - Gal4 / 1 - 119 trans - heterozygous mutant ( D ) or DIP - δ1 - 119 / + heterozygous ( C ) brains , which express MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ; green ) , in which DIP - δ - Gal4 either drives expression or UAS - DIP - α ( D ) or not ( C ) . Grayscale panels represent single channels , as indicated . The γ - lobe is outlined in white . ( E , F ) Confocal z - projections of adult dpr12Δ50 - 81 homozygous mutant brains , in which DIP - δ - Gal4 drives expression of either UAS - mtdT ( E ) or UAS - DIP - α - T2A - tdT ( F ; expected to induce expression of DIP - α as well as tdT encoded by a polycistronic message ) . In orange are longitudinal sections across the γ - lobe at the indicated location . The γ - lobe is outlined in white , as determined by FasII staining ( grey in E ' - F ' ) . Cyan is tdT within DIP - δ + cells . Grey boxes below the images describe the relevant components within PAM - DANs and γ - KCs .

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 O O O O O O O O O O O O O O O O O O O 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(C, D) Confocal z-projections of adult DIP-δT2A-Gal4/1-119 trans-heterozygous mutant (D) or DIP-δ1-119/+ heterozygous (C) brains, which express MiMIC mediated Dpr12GFSTF (Dpr12-GFP; green), in which DIP-δ-Gal4 either drives expression or UAS-DIP-α (D) or not (C). Grayscale panels represent single channels, as indicated. The γ-lobe is outlined in white.(E, F) Confocal z-projections of adult dpr12Δ50-81 homozygous mutant brains, in which DIP-δ-Gal4 drives expression of either UAS-mtdT (E) or UAS-DIP-α-T2A-tdT (F; expected to induce expression of DIP-α as well as tdT encoded by a polycistronic message). In orange are longitudinal sections across the γ-lobe at the indicated location. The γ-lobe is outlined in white, as determined by FasII staining (grey in E'-F'). Cyan is tdT within DIP-δ+ cells. Grey boxes below the images describe the relevant components within PAM-DANs and γ-KCs.


Figure 3 ( h ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in human , mouse , Japanese quail , painted turtle . YM . 254890 inhibits Ca2 + ion levels in cells transfected with GPR15 receptors of vertebrates . The fold - change in calcium levels was calculated based on the fluorescence intensity ( excitation and emission wavelengths : 490 and 520 nm , respectively ) . ( i , j ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in coelacanth , Asian bony tongue , and their mutants . ( k , l ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulating GPR15 in Japanese quail and its mutants . The fold - change in calcium levels was calculated based on the fluorescence intensity ( excitation and emission wavelengths : 490 and 520 nm , respectively ) .

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 O O O O O O O O O O O

Figure 3(h) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in human, mouse, Japanese quail, painted turtle. YM.254890 inhibits Ca2+ ion levels in cells transfected with GPR15 receptors of vertebrates. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).(i, j) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in coelacanth, Asian bony tongue, and their mutants. (k, l) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulating GPR15 in Japanese quail and its mutants. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).


Figure 4 ( a - h ) The migration of HEK293 cells expressing mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , and their mutants induced by ligand C10ORF99 . Data information : All data shown are generated from at least three technical replicates . Error bars indicate SD ( n = 3 ) . * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ( t - test ) . ( i - p ) The migration of CD4 + cells from GPR15 - knockout mice , engineered to express mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , and their mutants induced by ligand C10ORF99 . Data information : All data shown are generated from at least three technical replicates . Error bars indicate SD ( n = 3 ) . * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ( t - test ) . Ad - NC represents adeno - virus - based gene - delivery vector as negative control .

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 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

Figure 4(a-h) The migration of HEK293 cells expressing mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test).(i-p) The migration of CD4+ cells from GPR15-knockout mice, engineered to express mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test). Ad-NC represents adeno-virus-based gene-delivery vector as negative control.


Figure 5 ( a ) CCR9 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( b ) CCR9 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( c ) CCR9 expression in the small intestine ( SI ) and colon of Rana catesbiana Shaw and Rana nigromaculata . ( d ) GPR15 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( e ) GPR15 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( f ) C10ORF99 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( g ) C10ORF99 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( h ) GPR15 and C10ORF99 expression in the small intestine ( SI ) and colon of Rana catesbeiana Shaw and Rana nigromaculata . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) . ( i ) GPR15 expression in CD4 + T cell from chicken small intestine ( SI ) and colon . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) . ( j ) The migration of CD4 + cells from chicken small intestine ( SI ) and colon induced by ligand C10ORF99 . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 5(a) CCR9 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (b) CCR9 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (c) CCR9 expression in the small intestine (SI) and colon of Rana catesbiana Shaw and Rana nigromaculata. (d) GPR15 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (e) GPR15 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (f) C10ORF99 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (g) C10ORF99 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (h) GPR15 and C10ORF99 expression in the small intestine (SI) and colon of Rana catesbeiana Shaw and Rana nigromaculata. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).(i) GPR15 expression in CD4+ T cell from chicken small intestine (SI) and colon. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).(j) The migration of CD4+ cells from chicken small intestine (SI) and colon induced by ligand C10ORF99. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


Figure 6The expression levels of GPR15 , C10ORF99 , FOXP3 in mice fed with corn , chicken , and mixture of corn plus chicken , commercial food chow and a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled RNA from colon tissues from three mice . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) . The expression levels of IL - 10 in mice fed with corn , chicken , and mixture of corn plus chicken , commercial food chow and a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled RNA from colon tissues from three mice . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) . ( f , g ) SCFAs and acetic acid content in dietary experiments . Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) . ( h ) TNF - α and IL - 6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups . IL - 6 and TNF - α protein production levels , as determined by performing enzyme - linked immunosorbent assays . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) . ( i ) Luciferase activities of constructs with the mouse C10ORF99 promoter in HEK293 cells . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) . Luciferase activities of constructs with the Japanese quail C10ORF99 promoter in HEK293 cells . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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 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 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 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 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

Figure 6The expression levels of GPR15, C10ORF99, FOXP3 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).The expression levels of IL-10 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).(f, g) SCFAs and acetic acid content in dietary experiments. Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).(h) TNF-α and IL-6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups. IL-6 and TNF-α protein production levels, as determined by performing enzyme-linked immunosorbent assays. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).(i) Luciferase activities of constructs with the mouse C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).Luciferase activities of constructs with the Japanese quail C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


Figure 1 ( C ) BN - PAGE and SDS - PAGE assays with inactive and activated Rx - NRC2 . C - terminally 6xHA tagged Rx and C - terminally 4xMyc - tagged NRC2EEE were co - expressed with either free GFP or C - terminally GFP - tagged CP . Free mCherry - 4xMyc and mCherry - 6xHA fusions were used as controls in the treatments without NRC2 and Rx , respectively . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisks indicate bands corresponding to the activated NRC2 complex . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

Figure 1(C) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2. C-terminally 6xHA tagged Rx and C-terminally 4xMyc-tagged NRC2EEE were co-expressed with either free GFP or C-terminally GFP-tagged CP. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


Figure 2 ( B ) BN - PAGE and SDS - PAGE assays with inactive and activated Rx - NRC2 using different heavy and light sensor - helper combinations . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisk indicates bands corresponding to the activated NRC2 complex . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

Figure 2(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different heavy and light sensor-helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.