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 .

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

G. 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.


A . Fluorescent microscopy images showing that CRY2 , CIB1 and CO co - localized to the nucleus . Scale bars : 2

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

A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μ


B . 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 .

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

B. 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 .

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

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 .

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

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

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.


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

Confocal 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.


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

Confocal 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.


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

Confocal 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).


Confocal 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 .

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

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

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.


( 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 ) .

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

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

(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.


( 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 .

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(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.


Confocal 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 .

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Confocal 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 .

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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.


( 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 .

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(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 .

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(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.


( 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 ) .

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(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 ) .

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(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).


( 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 ) .

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

(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.


( 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 ) .

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(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 ) .

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(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 ) .

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(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).


The 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 ) .

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

The 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 ) .

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

The 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 ) .

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(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 ) .

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

(h) 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 ) .

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

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

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).


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

(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.


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

(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.


( B ) BN - PAGE and SDS - PAGE assays with inactive and activated Rx - NRC2 using different " heavy " and " light " 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 . A non - specific band was observed at ~ 110 kDa for the anti - FLAG antibody . Red asterisks indicate bands corresponding to the activated " heavy " NRC2 complexes . Blue asterisks indicate bands corresponding to " light " NRC2 complexes . Purple bands indicate intermediate molecular weight complexes combining " heavy " and " light " NRC2 . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different "heavy" and "light" 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. A non-specific band was observed at ~110 kDa for the anti-FLAG antibody. Red asterisks indicate bands corresponding to the activated "heavy" NRC2 complexes. Blue asterisks indicate bands corresponding to "light" NRC2 complexes. Purple bands indicate intermediate molecular weight complexes combining "heavy" and "light" NRC2. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( B ) BN - PAGE and SDS - PAGE assays with inactive and activated NRC - dependent sensors and NRC2 . 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 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC2. 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 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


BN - PAGE and SDS - PAGE assays with inactive and activated NRC - dependent sensors and NRC4AAA . Total protein was extracted with a HEPES - 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 NRC4 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC4AAA. Total protein was extracted with a HEPES-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 NRC4 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


C - terminally GFP - tagged NRC2EEE and C - terminally RFP - tagged Rx were co - expressed either with an EV - 4xMyc construct or a CP - 4xMyc construct in leaves of nrc2 / 3 / 4 N . benthamiana CRISPR mutant lines . ( A - B ) Single - plane confocal micrographs show the localization of both components of the inactive and active Rx - NRC2 system , together with PM marker RPW8 . 2 - BFP . Scale bars represent 10 µm . ( A ) NRC2EEE - GFP and Rx - RFP co - localize in the cytoplasm prior to activation . ( B ) Upon co - expression of CP and activation of the system , NRC2EEE forms puncta associated with the PM while Rx remains in the cytoplasm .

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

C-terminally GFP-tagged NRC2EEE and C-terminally RFP-tagged Rx were co-expressed either with an EV-4xMyc construct or a CP-4xMyc construct in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines. (A-B) Single-plane confocal micrographs show the localization of both components of the inactive and active Rx-NRC2 system, together with PM marker RPW8.2-BFP. Scale bars represent 10 µm. (A) NRC2EEE-GFP and Rx-RFP co-localize in the cytoplasm prior to activation. (B) Upon co-expression of CP and activation of the system, NRC2EEE forms puncta associated with the PM while Rx remains in the cytoplasm.


( C ) Membrane enrichment assays are consistent with microscopy , showing that inactive NRC2EEE - GFP is mostly present in the soluble ( cytoplasmic ) fraction , whereas activated NRC2EEE - GFP exhibits equal distribution across soluble and membrane fractions . Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP . T = total , S = soluble , M = membrane . ATPase was used as a membrane marker . Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining ( PS ) . Red asterisks indicate bands matching the expected MW for each protein . The experiment was repeated two times .

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

(C) Membrane enrichment assays are consistent with microscopy, showing that inactive NRC2EEE-GFP is mostly present in the soluble (cytoplasmic) fraction, whereas activated NRC2EEE-GFP exhibits equal distribution across soluble and membrane fractions. Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP. T = total, S = soluble, M = membrane. ATPase was used as a membrane marker. Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining (PS). Red asterisks indicate bands matching the expected MW for each protein. The experiment was repeated two times.


( B ) BN - PAGE and SDS - PAGE assays with infected and uninfected leaves expressing Rx and NRC2 . 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 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with infected and uninfected leaves expressing Rx and NRC2. 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 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( A - B ) Single - plane confocal micrographs show the localization of NRC2 together with Rx or free RFP during PVX infection . C - terminally GFP - tagged NRC2EEE was co - infiltrated with either EV - RFP or Rx - RFP in leaves of nrc2 / 3 / 4 N . benthamiana CRISPR mutant lines and infected with PVX by agroinfection . Scale bars represent 10 µm . ( A ) In the absence of Rx , NRC2EEE - GFP is localized to the cytoplasm during PVX infection . ( B ) When Rx is present , NRC2EEE forms puncta associated with the PM during PVX infection .

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

(A-B) Single-plane confocal micrographs show the localization of NRC2 together with Rx or free RFP during PVX infection. C-terminally GFP-tagged NRC2EEE was co-infiltrated with either EV-RFP or Rx-RFP in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines and infected with PVX by agroinfection. Scale bars represent 10 µm. (A) In the absence of Rx, NRC2EEE-GFP is localized to the cytoplasm during PVX infection. (B) When Rx is present, NRC2EEE forms puncta associated with the PM during PVX infection.


( A ) MEFs were transfected with GFP - tagged wild - type ( WT ) or mutant Parkin expression plasmid followed by an 18 - h treatment of CCCP . Cells are immunostained with a Tom20 antibody to visualize mitochondria ( red ) . GFP - parkin - transfected cells are marked by dotted lines . Arrows indicate parkin - positive mitochondria or mitochondrial aggregates . Bar , 10 µm . ( B ) The average percentages of mitochondria - free cells from three independent experiments from A are presented with standard deviation as error bar . * * , P < 0 .

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

(A) MEFs were transfected with GFP-tagged wild-type (WT) or mutant Parkin expression plasmid followed by an 18-h treatment of CCCP. Cells are immunostained with a Tom20 antibody to visualize mitochondria (red). GFP-parkin-transfected cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of mitochondria-free cells from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01


( C ) MEFs were transfected and treated with CCCP as described in A , followed by an immunoblotting analysis with antibodies for cytochrome c , actin , and parkin . Note that levels of parkin R275W and R42P mutant were lower , as previously reported ( Wang et al . , 2005 ) .

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

(C) MEFs were transfected and treated with CCCP as described in A, followed by an immunoblotting analysis with antibodies for cytochrome c, actin, and parkin. Note that levels of parkin R275W and R42P mutant were lower, as previously reported (Wang et al., 2005).


CCCP - induced parkin - mitochondrial aggregate formation is an intermediate step for mitophagy . ( A ) MEFs expressing WT GFP - parkin were treated with CCCP or CCCP and nocodazole ( NOC , 10 µM ) for 8 , 16 , and 24 h as indicated . Cells were immunostained with anti - Tom20 to visualize mitochondria . GFP - parkin - positive cells are marked by dotted lines . Arrows indicate parkin - positive mitochondria or mitochondrial aggregates . Bar , 25 µm . ( B ) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar . * * , P < 0 . 01 .

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

CCCP-induced parkin-mitochondrial aggregate formation is an intermediate step for mitophagy. (A) MEFs expressing WT GFP-parkin were treated with CCCP or CCCP and nocodazole (NOC, 10 µM) for 8, 16, and 24 h as indicated. Cells were immunostained with anti-Tom20 to visualize mitochondria. GFP-parkin-positive cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 25 µm. (B) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01.


( A ) MEFs were transfected with GFP wild - type ( WT ) , A240R , and T415N mutant parkin followed by CCCP treatment for 8 h . Cells were immunostained with antibodies for cytochrome c ( red ) and ubiquitin ( blue ) . Arrows indicate mitochondrial aggregates . Bar , 10 µm . ( B ) The average percentages of ubiquitin - positive mitochondrial aggregates from three independent experiments from A are presented with standard deviation as error bar .

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

(A) MEFs were transfected with GFP wild-type (WT), A240R, and T415N mutant parkin followed by CCCP treatment for 8 h. Cells were immunostained with antibodies for cytochrome c (red) and ubiquitin (blue). Arrows indicate mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of ubiquitin-positive mitochondrial aggregates from three independent experiments from A are presented with standard deviation as error bar.


( C and D ) MEFs were transfected and treated with CCCP as described in A . Cells were subjected to the fractionation to isolate crude mitochondria . Cytoplasmic and mitochondrial fractions were subjected to immunoblotting analysis using antibodies for ubiquitin , parkin , cytochrome c , and cytosolic Hsp90 .

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

(C and D) MEFs were transfected and treated with CCCP as described in A. Cells were subjected to the fractionation to isolate crude mitochondria. Cytoplasmic and mitochondrial fractions were subjected to immunoblotting analysis using antibodies for ubiquitin, parkin, cytochrome c, and cytosolic Hsp90.


Parkin - mediated ubiquitination recruits p62 and HDAC6 . MEFs were transfected with WT , A240R , and T415N mutant GFP - parkin followed by CCCP treatment for 8 h . Cells were double immunostained with cytochrome c ( red ) and p62 antibody ( blue ) in A , and cytochrome c ( red ) and HDAC6 antibody in C . Arrows indicate mitochondrial aggregates . ( B and D ) The average percentages of p62 - or HDAC6 - positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar .

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

Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar.


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


( E ) Wild - type and HDAC6 knockout ( KO ) MEFs were transfected with parkin - GFP or cotransfected with a Flag - tagged HDAC6 followed by CCCP treatment for 16 h as indicated . Cells are immunostained with Tom20 ( red ) and Flag ( blue ) antibodies . Bar , 25 µm .

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

(E) Wild-type and HDAC6 knockout (KO) MEFs were transfected with parkin-GFP or cotransfected with a Flag-tagged HDAC6 followed by CCCP treatment for 16 h as indicated. Cells are immunostained with Tom20 (red) and Flag (blue) antibodies. Bar, 25 µm.


( F ) Wild - type MEFs were transfected with control or HDAC6 siRNA and parkin - GFP , and treated with or without CCCP for 16 h . Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20 , parkin , HDAC6 , and actin .

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

(F) Wild-type MEFs were transfected with control or HDAC6 siRNA and parkin-GFP, and treated with or without CCCP for 16 h. Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20, parkin, HDAC6, and actin.


MEFs were transfected with control or cortactin RNAi and parkin - GFP , and incubated with or without CCCP for 16 h as indicated . ( A ) Cells were subjected to immunostaining with antibodies for Tom20 and parkin . Bar , 10 µm .

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

MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated. (A) Cells were subjected to immunostaining with antibodies for Tom20 and parkin. Bar, 10 µm.


MEFs were transfected with control or cortactin RNAi and parkin - GFP , and incubated with or without CCCP for 16 h as indicated . ( B ) Cell lysates were subjected to immunoblotting with antibodies for Tom20 , parkin , cortactin , and actin .

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

MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated.(B) Cell lysates were subjected to immunoblotting with antibodies for Tom20, parkin, cortactin, and actin.


( b ) A53T α - synuclein ( α - syn ) expression was induced in stable PC12 cells for 48 h , then switched off for 24 h . Cells were treated with DMSO ( control ) , or with 0 . 94 μM , 4 . 7 μM or 47 μM of SMER10 ; 0 . 86 μM , 4 . 3 μM or 43 μM of SMER18 ; or 0 . 9 μM , 4 . 7 μM or 47 μM of SMER28 added in the switch - off period . A53T α - syn levels were analyzed by immunoblotting with antibody against HA ( left ) and densitometry analysis relative to actin ( right ) . Error bars denote s . e . m . P = 0 . 0917 , P = 0 . 009 , P = 0 . 0001 ( for increasing concentrations of SMER10 ) ; P = 0 . 0068 , P = 0 . 0023 , P = 0 . 0002 ( for increasing concentrations of SMER18 ) ; P = 0 . 0016 , P 0 . 0001 , P 0 . 0001 ( for increasing concentrations of SMER28 ) .

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

(b) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28).


( c ) COS - 7 cells transfected with EGFP - HDQ74 construct were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( Rap ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , and we assessed the proportions of apoptotic or aggregate - containing transfected cells . Error bars : 95 % confidence interval . P 0 . 0001 ( Rap and SMER28 ) , P = 0 . 013 ( SMER10 ) , P = 0 . 019 ( SMER18 ) ( aggregation ) ; P 0 . 0001 ( Rap , SMER18 and SMER28 ) , P = 0 . 004 ( SMER10 ) ( cell death ) .

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

(c) COS-7 cells transfected with EGFP-HDQ74 construct were treated with DMSO (control), 0.2 μM rapamycin (Rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, and we assessed the proportions of apoptotic or aggregate-containing transfected cells. Error bars: 95% confidence interval. P 0.0001 (Rap and SMER28), P = 0.013 (SMER10), P = 0.019 (SMER18) (aggregation); P 0.0001 (Rap, SMER18 and SMER28), P = 0.004 (SMER10) (cell death).


( d ) Wild - type ( Atg5 + / + ) and knockout ( Atg5 − / − ) Atg5 MEFs were transfected with EGFP - HDQ74 and treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h , and we assessed the percentages of aggregate - containing transfected cells . The control ( DMSO - treated ) values were fixed at 1 for both cell lines . Error bars : 95 % confidence interval . P = 0 . 039 ( SMER10 ) , P 0 . 0001 ( SMER18 and SMER28 ) ( in Atg5 + / + cells ) ; P = 0 . 092 ( SMER10 ) , P = 0 . 271 ( SMER18 ) , P = 0 . 358 ( SMER28 ) ( in Atg5 − / − cells ) . Note that EGFP - HDQ74 aggregation was higher in Atg5 − / − than in Atg5 + / + cells ( Supplementary Fig . 3a ) .

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

(d) Wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs were transfected with EGFP-HDQ74 and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h, and we assessed the percentages of aggregate-containing transfected cells. The control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95% confidence interval. P = 0.039 (SMER10), P 0.0001 (SMER18 and SMER28) (in Atg5+/+ cells); P = 0.092 (SMER10), P = 0.271 (SMER18), P = 0.358 (SMER28) (in Atg5−/− cells). Note that EGFP-HDQ74 aggregation was higher in Atg5−/− than in Atg5+/+ cells (Supplementary Fig. 3a).


e ) The percentages of EGFP - positive COS - 7 cells with EGFP - HDQ74 aggregates as in c , treated with DMSO ( control ) , 10 μM lactacystin ( proteasome inhibitor ) or both 10 μM lactacystin along with either 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , expressed as odds ratios . Error bars : 95 % confidence interval . P 0 . 0001 ( control versus Lact ) , P = 0 . 014 ( SMER10 versus Lact ) , P 0 . 0001 ( SMER18 versus Lact ) , P = 0 . 001 ( SMER28 versus Lact ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

e) The percentages of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor) or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, expressed as odds ratios. Error bars: 95% confidence interval. P 0.0001 (control versus Lact), P = 0.014 (SMER10 versus Lact), P 0.0001 (SMER18 versus Lact), P = 0.001 (SMER28 versus Lact). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) COS - 7 cells transfected with EGFP - LC3 construct for 4 h were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( positive control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 16 h , and analyzed by fluorescence microscopy . The effects of treatment on the percentage of EGFP - positive cells with > 5 EGFP - LC3 vesicles are shown . Error bars denote s . e . m . P 0 . 0001 ( all SMERs ) .

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

(a) COS-7 cells transfected with EGFP-LC3 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (positive control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 16 h, and analyzed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. Error bars denote s.e.m. P 0.0001 (all SMERs).


( b ) HeLa cells stably expressing EGFP - LC3 were treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h . Confocal sections show cells containing EGFP - positive autophagic vesicles . Nuclei are stained with DAPI . Bar , 20 μM .

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

(b) HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM.


( c ) HeLa cells stably expressing EGFP - LC3 were treated for 4 h with DMSO ( control ) or 200 nM bafilomycin A1 ( Baf ) , or with 200 nM bafilomycin A1 and 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 . Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1 . Levels of EGFP - LC3 - II were determined by immunoblotting with antibody against EGFP ( above ) and densitometry analysis relative to actin ( below ) . Error bars denote s . e . m . P = 0 . 0259 ( baf ) , P 0 . 0001 ( SMER10 ) , P = 0 . 0003 ( SMER18 and SMER28 ) versus control ; P = 0 . 0025 ( SMER10 ) , P = 0 . 0218 ( SMER18 ) , P = 0 . 0195 ( SMER28 ) versus bafilomycin A1 . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

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

(c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (Baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (above) and densitometry analysis relative to actin (below). Error bars denote s.e.m. P = 0.0259 (baf), P 0.0001 (SMER10), P = 0.0003 (SMER18 and SMER28) versus control; P = 0.0025 (SMER10), P = 0.0218 (SMER18), P = 0.0195 (SMER28) versus bafilomycin A1. ***P 0.001; **P 0.01; *P 0.05.


( a - c ) Flies treated with 100 μM SMER10 ( a ) , 200 μM SMER18 ( b ) or 100 μM SMER28 ( c ) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO ( control ) alone ( 2 d after eclosion ) . Rhabdomere counts from all three independent experiments are included . n = 600 ommatidia ( SMER10 ) , n = 1 , 500 ommatidia ( SMER18 ) and n = 600 ommatidia ( SMER28 ) . Mann - Whitney test values P 0 . 0001 ( all SMERs ) . Student ' s t - test ( 1 - tailed ) P = 0 . 005 ( SMER10 ) , P = 0 . 004 ( SMER18 ) , P = 0 . 03 ( SMER28 ) comparing means of distributions from independent experiments . These SMER concentrations cause no overt toxicity to flies ( see Supplementary Methods ) . Distributions of DMSO - treated flies may vary when SMERs are treated in different experiments at different times . For instance , an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time - dependent .

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

(a-c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO (control) alone (2 d after eclosion). Rhabdomere counts from all three independent experiments are included. n = 600 ommatidia (SMER10), n = 1,500 ommatidia (SMER18) and n = 600 ommatidia (SMER28). Mann-Whitney test values P 0.0001 (all SMERs). Student's t-test (1-tailed) P = 0.005 (SMER10), P = 0.004 (SMER18), P = 0.03 (SMER28) comparing means of distributions from independent experiments. These SMER concentrations cause no overt toxicity to flies (see Supplementary Methods). Distributions of DMSO-treated flies may vary when SMERs are treated in different experiments at different times. For instance, an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time-dependent.


( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( a ) , 43 μM SMER18 ( b ) or 47 μM SMER28 ( c ) ) or both for the 8 h switch - off period - was analyzed by immunoblotting with antibody against HA ( above ) and densitometry analysis relative to actin ( below ) . The concentration of rapamycin is saturating for its effect on the clearance of A53T α - synuclein . Error bars denote s . e . m . P = 0 . 0025 ( Rap ) , P = 0 . 0018 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 0001 ( Rap or SMER10 versus SMER10 + Rap ) ( a ) ; P = 0 . 0069 ( Rap ) , P = 0 . 0498 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 0038 ( Rap versus SMER18 + Rap ) , P = 0 . 0007 ( SMER18 versus SMER18 + Rap ) ( b ) ; P 0 . 0001 ( Rap , SMER28 , Rap versus SMER28 + Rap , SMER28 versus SMER28 + Rap ) ( c ) .

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

(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (a), 43 μM SMER18 (b) or 47 μM SMER28 (c)) or both for the 8 h switch-off period-was analyzed by immunoblotting with antibody against HA (above) and densitometry analysis relative to actin (below). The concentration of rapamycin is saturating for its effect on the clearance of A53T α-synuclein. Error bars denote s.e.m. P = 0.0025 (Rap), P = 0.0018 (SMER10), P 0.0001 (SMER10 + Rap), P 0.0001 (Rap or SMER10 versus SMER10 + Rap) (a); P = 0.0069 (Rap), P = 0.0498 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.0038 (Rap versus SMER18 + Rap), P = 0.0007 (SMER18 versus SMER18 + Rap) (b); P 0.0001 (Rap, SMER28, Rap versus SMER28 + Rap, SMER28 versus SMER28 + Rap) (c).


( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates ( above ) and cell death ( below ) in COS - 7 cells , as in Figure 1c - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( d ) , 43 μM SMER18 ( e ) or 47 μM SMER28 ( f ) ) or both for 24 h - were expressed as odds ratios . Error bars : 95 % confidence interval . Aggregation ( top panels ) : P = 0 . 248 ( Rap ) , P = 0 . 217 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 001 ( Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 248 ( Rap ) , P = 0 . 543 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 008 ( Rap versus SMER18 + Rap ) , P = 0 . 002 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 248 ( Rap ) , P = 0 . 002 ( SMER28 ) , P 0 . 0001 ( SMER28 + Rap ) , P 0 . 0001 ( Rap versus SMER28 + Rap ) , P = 0 . 012 ( SMER28 versus SMER28 + Rap ) ( f ) . Cell death ( bottom panels ) : P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER10 , SMER10 + Rap , Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 002 ( Rap ) , P = 0 . 948 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 015 ( Rap versus SMER18 + Rap ) , P 0 . 0001 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER28 , SMER28 + Rap , Rap or SMER28 versus SMER28 + Rap ) ( f ) . Note that we have treated cells for a shorter time in this experiment ( 24 h ) , compared with Figure 1c ( 48 h ) . This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (above) and cell death (below) in COS-7 cells, as in Figure 1c-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)) or both for 24 h-were expressed as odds ratios. Error bars: 95% confidence interval. Aggregation (top panels): P = 0.248 (Rap), P = 0.217 (SMER10), P 0.0001 (SMER10 + Rap), P 0.001 (Rap or SMER10 versus SMER10 + Rap) (d); P = 0.248 (Rap), P = 0.543 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.008 (Rap versus SMER18 + Rap), P = 0.002 (SMER18 versus SMER18 + Rap) (e); P = 0.248 (Rap), P = 0.002 (SMER28), P 0.0001 (SMER28 + Rap), P 0.0001 (Rap versus SMER28 + Rap), P = 0.012 (SMER28 versus SMER28 + Rap) (f). Cell death (bottom panels): P = 0.002 (Rap), P 0.0001 (SMER10, SMER10 + Rap, Rap or SMER10 versus SMER10 + Rap) (d); P = 0.002 (Rap), P = 0.948 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.015 (Rap versus SMER18 + Rap), P 0.0001 (SMER18 versus SMER18 + Rap) (e); P = 0.002 (Rap), P 0.0001 (SMER28, SMER28 + Rap, Rap or SMER28 versus SMER28 + Rap) (f). Note that we have treated cells for a shorter time in this experiment (24 h), compared with Figure 1c (48 h). This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated for 24 h with either DMSO ( control ) , or with 47 μM SMER10 and its analogs ( SMER10a - c ) ( a ) , 43 μM SMER18 and its analogs ( SMER18a - l ) ( b ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( c ) - was analyzed by immunoblotting with anti - HA antibody ( above ) and densitometry analysis relative to actin ( below ) . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars denote s . e . m . P = 0 . 0058 ( SMER10a ) , P = 0 . 6736 ( SMER10b ) , P = 0 . 9507 ( SMER10c ) , P = 0 . 0481 ( SMER10 ) ( a ) ; P = 0 . 0006 ( SMER18a ) , P = 0 . 0249 ( SMER18b ) , P = 0 . 0167 ( SMER18c ) , P = 0 . 0117 ( SMER18d ) , P = 0 . 0269 ( SMER18e ) , P = 0 . 0165 ( SMER18f ) , P = 0 . 0148 ( SMER18g ) , P = 0 . 0011 ( SMER18h ) , P = 0 . 7369 ( SMER18i ) , P = 0 . 0012 ( SMER18j ) , P = 0 . 1531 ( SMER18k ) , P = 0 . 0006 ( SMER18l ) , P = 0 . 0001 ( SMER18 ) ( b ) ; P = 0 . 0014 ( SMER28a ) , P = 0 . 0002 ( SMER28b ) , P = 0 . 0001 ( SMER28d ) , P = 0 . 0048 ( SMER28h ) , P = 0 . 0002 ( SMER28i ) , P = 0 . 0162 ( SMER28j ) , P 0 . 0001 ( SMER28c , SMER28e - g , SMER28k , SMER28 ) ( c ) .

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

(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c)-was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below). All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars denote s.e.m. P = 0.0058 (SMER10a), P = 0.6736 (SMER10b), P = 0.9507 (SMER10c), P = 0.0481 (SMER10) (a); P = 0.0006 (SMER18a), P = 0.0249 (SMER18b), P = 0.0167 (SMER18c), P = 0.0117 (SMER18d), P = 0.0269 (SMER18e), P = 0.0165 (SMER18f), P = 0.0148 (SMER18g), P = 0.0011 (SMER18h), P = 0.7369 (SMER18i), P = 0.0012 (SMER18j), P = 0.1531 (SMER18k), P = 0.0006 (SMER18l), P = 0.0001 (SMER18) (b); P = 0.0014 (SMER28a), P = 0.0002 (SMER28b), P = 0.0001 (SMER28d), P = 0.0048 (SMER28h), P = 0.0002 (SMER28i), P = 0.0162 (SMER28j), P 0.0001 (SMER28c, SMER28e-g, SMER28k, SMER28) (c).


( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates in COS - 7 cells as in Figure 1c - treated for 48 h with either DMSO ( control ) , or with 47 μM SMER10 and its analog ( SMER10a ) ( d ) , 43 μM SMER18 and its analogs ( SMER18a , SMER18c - h , SMER18j , SMER18l ) ( e ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( f ) - were expressed as odds ratios . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars : 95 % confidence interval . P = 0 . 003 ( SMER10a ) , P = 0 . 004 ( SMER10 ) ( d ) ; P 0 . 0001 ( SMER18a , SMER18c , SMER18d , SMER18f , SMER18h ) , P = 0 . 009 ( SMER18e ) , P = 0 . 001 ( SMER18g ) , P = 0 . 382 ( SMER18j ) , P = 0 . 067 ( SMER18l ) , P = 0 . 031 ( SMER18 ) ( e ) ; P 0 . 0001 ( SMER28a , SMER28c , SMER28e - g , SMER28i - k , SMER28 ) , P = 0 . 574 ( SMER28b ) , P = 0 . 041 ( SMER28d ) , P = 0 . 002 ( SMER28h ) ( f ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


B . RIBEYE mutations do not alter mouse survival . Data show surviving post - weaning mice ( at > P21 ) derived from matings between mice heterozygous for the RBEKI allele ( dark red ) or the RBEKO allele ( bright red ) normalized for the total number of mice ( absolute numbers per genotype / absolute number of all mice per respective breeding strategy ) The expected Mendelian ratio is indicated by the dashed line . Statistical analysis using the 2 - test revealed no statistical difference between wild - type and mutant mice ( RBEKI allele p = 0 . 33 ; RBEKO allele p = 0 . 11 ) .

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

B. RIBEYE mutations do not alter mouse survival. Data show surviving post-weaning mice (at >P21) derived from matings between mice heterozygous for the RBEKI allele (dark red) or the RBEKO allele (bright red) normalized for the total number of mice (absolute numbers per genotype/absolute number of all mice per respective breeding strategy) The expected Mendelian ratio is indicated by the dashed line. Statistical analysis using the 2-test revealed no statistical difference between wild-type and mutant mice (RBEKI allele p=0.33; RBEKO allele p=0.11).


C . Representative immunoblot of retina homogenates from homozygous RBEWT / WT , RBEKI / KI and RBEKO / KO mice probed with antibodies to CtBP2 ( to detect the B - domain of RIBEYE and CtBP2 ) and to EGFP ( to detect GCamP3 in the RIBEYE A - domain GCamP3 fusion protein referred to as RIB - G3 ) .

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

C. Representative immunoblot of retina homogenates from homozygous RBEWT/WT, RBEKI/KI and RBEKO/KO mice probed with antibodies to CtBP2 (to detect the B-domain of RIBEYE and CtBP2) and to EGFP (to detect GCamP3 in the RIBEYE A-domain GCamP3 fusion protein referred to as RIB-G3).