B . Yeast two - hybrid of interactions between FREE1 microprocessor . SD - 2 : SD / - Leu - Trp . SD - 4 : SD / - Ade - His - Leu - Trp . Three dots represent three yeast colonies randomly picked out in each sample .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Yeast two-hybrid analysis of the interactions between FREE1 and core microprocessor. SD-2: SD/-Leu-Trp. SD-4: SD/-Ade-His-Leu-Trp. Three dots represent three yeast colonies randomly picked out in each sample.


C . BiFC to verify interaction between FREE1 CPL1 . YC - FREE1 YN - CPL1 , were co - expressed with mCherry - HYL1 , SE - mCherry , or DCL1 - mCherry in Arabidopsis protoplasts , respectively , followed by confocal observation .

O O O O O O O O O O O O O O O O O O O O O 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. BiFC assay to verify the interaction between FREE1 and CPL1. YC-FREE1 and YN-CPL1, were co-expressed with mCherry-HYL1, SE-mCherry, or DCL1-mCherry in Arabidopsis protoplasts, respectively, followed by confocal observation.


D . Co - immunoprecipitation of interactions between FREE1 CPL1 CPL2 . Arabidopsis protoplasts co - expressing mCherry - FREE1 with YFP , YFP - CPL1 or YFP - CPL2 were subjected to extraction IP with GFP - trap followed by immunoblotting with mCherry GFP antibodies .

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

D. Co-immunoprecipitation analysis of interactions between FREE1 and CPL1 and CPL2. Arabidopsis protoplasts co-expressing mCherry-FREE1 with YFP, YFP-CPL1 or YFP-CPL2 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with mCherry and GFP antibodies.


E . Co - immunoprecipitation of associations between FREE1 microprocessor components . Transgenic expressing GFP - FREE1 were subjected to extraction IP with GFP - trap followed by immunoblotting with indicated antibodies .

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

E. Co-immunoprecipitation analysis of the associations between FREE1 and microprocessor components. Transgenic plants expressing GFP-FREE1 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with indicated antibodies.


B . Small gel blots for 7 - day - old seedlings Col - 0 free1 - ctmut . U6 was used as a loading control . numbers indicate relative abundance of miR168 , miR166 , miR162 , siRNA1003 , tasiRNA255 , IR71 , miR156 miR172 in ( in WT was arbitrarily annotated as 1 . 00 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Small RNA gel blots for 7-day-old seedlings Col-0 and free1-ctmut plants. U6 was used as a loading control. The numbers indicate the relative abundance of miR168, miR166, miR162, siRNA1003, tasiRNA255, IR71, miR156 and miR172 in the plants (the intensity in WT was arbitrarily annotated as 1.00).


A . Detection of HYL1 phosphorylation in Col - 0 , free1 - ctmut or cpl1 - 8 . extracted from 7 - day - old Arabidopsis seedlings were separated by Phos - tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies . numbers in first row indicate relative grayscale intensities of total HYL1 , numbers in second row indicate relative intensities of dephosphorylated HYL1 phosphorylated HYL1 , in WT was arbitrarily set to 1 . 00 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Detection of HYL1 phosphorylation in Col-0, free1-ctmut or cpl1-8. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies. The numbers in the first row indicate the relative grayscale intensities of total HYL1, the numbers in the second row indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the intensity in WT was arbitrarily set to 1.00.


B . Transgenic expressing pHYL1 : : YFP - HYL1 in Col - 0 , free1 - ctmut or cpl1 - 8 backgrounds were subjected to extraction IP with GFP - trap , followed by detection with phos - tag BTL - 111 or GFP antibody . numbers indicate relative intensities of each band detected with phos - tag BTL - 111 anti - GFP , first lane was arbitrarily set to 1 . 00 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transgenic plants expressing pHYL1:: YFP-HYL1 in Col-0, free1-ctmut or cpl1-8 backgrounds were subjected to protein extraction and IP with GFP-trap, followed by detection with phos-tag BTL-111 or GFP antibody. The numbers indicate the relative intensities of each band detected with phos-tag BTL-111 and anti-GFP, and the first lane was arbitrarily set to 1.00.


C . Confocal observation of YFP - HYL1 - containing D - bodies in Col - 0 free1 - ctmut roots . Arrows indicated YFP - HYL1 - containing D - bodies . D . Statistically of number of YFP - HYL1 - containing D - bodies as shown in Fig 4C . X - axis represents number of D - bodies per , Y - axis represents of with corresponding numbers . was performed by observing more than 400 from 20 roots for each

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal observation of the YFP-HYL1-containing D-bodies in Col-0 and free1-ctmut roots. Arrows indicated the YFP-HYL1-containing D-bodies. D. Statistically analysis of the number of YFP-HYL1-containing D-bodies as shown in Fig 4C. The X-axis represents the number of D-bodies per cell, and the Y-axis represents the percentage of cells with the corresponding numbers. The quantification was performed by observing more than 400 cells from 20 roots for each genotype


E . abundance of components of miRNA processing in Col - 0 free1 - ctmut . extracted from 7 - day - old Arabidopsis seedlings were used for immunoblotting with indicated antibodies . numbers represent intensities of each band detected with indicated antibody relative to anti - UGPase , first lane was arbitrarily set to 1 . 00 .

E. Protein abundance of core components of miRNA processing complex in Col-0 and free1-ctmut. Proteins extracted from 7-day-old Arabidopsis seedlings were used for immunoblotting with indicated antibodies. The numbers represent the intensities of each band detected with indicated antibody relative to anti-UGPase, and the first lane was arbitrarily set to 1.00.


F . extracted from 7 - day - old Arabidopsis seedlings treated with CHX ( 0 . 5 mM ) in for 0h , 2h , 4h were used for immunoblotting with indicated antibodies . numbers represent relative intensities of each band detected with anti - HYL1 anti - Actin , first lane was arbitrarily set to 1 . 00 .

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

F. Proteins extracted from 7-day-old Arabidopsis seedlings treated with CHX (0.5 mM) in dark for 0h, 2h, 4h were used for immunoblotting analysis with indicated antibodies. The numbers represent the relative intensities of each band detected with anti-HYL1 and anti-Actin, and the first lane was arbitrarily set to 1.00.


G . extracted from 7 - day - old Arabidopsis seedlings with 3 - d continuous or treatment were used for immunoblotting with indicated antibodies .

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

G. Proteins extracted from 7-day-old Arabidopsis seedlings with 3-d continuous light or dark treatment were used for immunoblotting analysis with indicated antibodies.


A . extracted from Arabidopsis protoplasts co - expressing YFP - CPL1 different amount of 3myc - FREE1 were used for immunoprecipitation with GFP - Trap followed by detection with indicated antibodies .

A. Proteins extracted from Arabidopsis protoplasts co-expressing YFP-CPL1 and different amount of 3myc-FREE1 were used for immunoprecipitation with GFP-Trap followed by detection with indicated antibodies.


B . extracted from transgenic expressing YFP - CPL1 in Col - 0 or free1 - ctmut background were used for IP with GFP - trap followed by immunoblotting with indicated antibodies .

B. Proteins extracted from transgenic plants expressing YFP-CPL1 in Col-0 or free1-ctmut background were used for IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.


C . Statistically of relative of FREE1 , HYL1 SE in Col - 0 or free1 - ctmut background ( IP / Input ) . Data are presented as means ± s . d . ( 3 biological replicates ) , asterisk indicates significant difference between samples ( * * P < 0 . 01 by Student ' s t - , compared with WT ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Statistically analysis of the relative intensity of FREE1, HYL1 and SE in Col-0 or free1-ctmut background (IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).


D . Transgenic of YFP - HYL1 / WT , YFP - HYL1 / free1 - ctmut , YFP - HYL1 / cpl1 - 8 GFP / WT were subjected to extraction IP with GFP - trap followed by immunoblotting with indicated antibodies .

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

D. Transgenic plants of YFP-HYL1 / WT, YFP-HYL1 / free1-ctmut, YFP-HYL1 / cpl1-8 and GFP / WT were subjected to protein extraction and IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.


E . Statistically analyzed relative of FREE1 HYL1 ( IP / Input ) . Data are presented as means ± s . d . ( 3 biological replicates ) , asterisk indicates significant difference between samples ( * * P < 0 . 01 by Student ' s t - , compared with WT ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Statistically analyzed the relative intensity of FREE1 and HYL1(IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).


F . extracted from 7 - day - old Arabidopsis seedlings were separated by Phos - tag polyacrylamide gels followed by immunoblotting with HYL1 antibody . numbers indicate relative intensities of dephosphorylated HYL1 phosphorylated HYL1 , first lane was arbitrarily set to 1 . 00 .

O O O O O O O O O O O O O O O O O O O O O O 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. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels followed by immunoblotting analysis with HYL1 antibody. The numbers indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the first lane was arbitrarily set to 1.00.


( A ) Mass spectrometry ( MS ) of indicated electrophoretic bands in Coomassie blue ( left panel ) , pulled down by Flag - tagged SIRT5 from 293T were indicated on right .

O O O O O O O O O O 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) Mass spectrometry (MS) assay of indicated electrophoretic bands in Coomassie blue staining (left panel), and proteins pulled down by Flag-tagged SIRT5 from 293T cells were indicated on the right.


( B ) Lysates from 293T transfected with HA - TRIM21 Flag - SIRT5 or vector control ( - ) as indicated were immunoprecipitated ( IP ) using anti - Flag antibody , bound were analyzed by western blotting ( WB ) . Data information : IP WB data in this work otherwise indicated are representative of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysates from 293T cells transfected with HA-TRIM21 and Flag-SIRT5 or vector control (-) as indicated were immunoprecipitated (IP) using anti-Flag antibody, and bound proteins were analyzed by western blotting (WB). Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( C , Lysates from BMDMs were immunoprecipitated with anti - SIRT5 ( C ) or control IgG antibodies as indicated then analyzed by western blotting . Data information : IP WB data in this work otherwise indicated are representative of at least three independent experiments .

(C, Lysates from BMDMs were immunoprecipitated with anti-SIRT5 (C) or control IgG antibodies as indicated and then analyzed by western blotting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


D ) Lysates from BMDMs were immunoprecipitated with anti - TRIM21 ( D ) or control IgG antibodies as indicated then analyzed by western blotting . Data information : IP WB data in this work otherwise indicated are representative of at least three independent experiments .

D) Lysates from BMDMs were immunoprecipitated with anti-TRIM21 (D) or control IgG antibodies as indicated and then analyzed by western blotting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( E ) Bacterially expressed purified Flag - SIRT5 were incubated with purified HA - TRIM21 prior to pull - down with anti - HA antibody followed by western blot . Data information : WB data in this work otherwise indicated are representative of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterially expressed and purified Flag-SIRT5 were incubated with purified HA-TRIM21 proteins prior to pull-down with anti-HA antibody followed by western blot analysis. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( F ) Immunoblot ( IB ) of whole - lysates ( WCLs ) immunoprecipitates ( IPs ) derived from 293T transfected with Flag - SIRT5 together with HA - TRIM21 or indicated HA - tagged mutant TRIM21 constructs . were treated with 10 μM MG132 for 4 hours before harvesting . HA , hemagglutinin .

(F) Immunoblot (IB) analysis of whole-cell lysates (WCLs) and immunoprecipitates (IPs) derived from 293T cells transfected with Flag-SIRT5 together with HA-TRIM21 or indicated HA-tagged mutant TRIM21 constructs. Cells were treated with 10 μM MG132 for 4 hours before harvesting. HA, hemagglutinin.


( G ) IB of WCLs derived from 293T transfected with increasing amounts of Flag - TRIM21 construct . Where indicated , 10 μM MG132 was added for 4 hours before harvesting .

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

(G) IB analysis of WCLs derived from 293T cells transfected with increasing amounts of Flag-TRIM21 construct. Where indicated, 10 μM MG132 was added for 4 hours before harvesting.


( H ) 293T transfected with control or TRIM21 siRNA for 48 hours were then treated with 100 ng / mL CHX for indicated time period before harvesting . expression was analyzed by western blotting ( left panel ) . SIRT5 abundance was quantified by ImageJ plotted as indicated ( right panel ) . Data information : WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(H) 293T cells transfected with control or TRIM21 siRNA for 48 hours were then treated with 100 ng/mL CHX for the indicated time period before harvesting. Protein expression was analyzed by western blotting (left panel). The SIRT5 protein abundance was quantified by ImageJ and plotted as indicated (right panel). Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( I ) IB of WCLs derived from TRIM21 + / + TRIM21 - / - BMDMs treated with 100 ng / ml CHX for indicated time period before harvesting ( left panel ) . SIRT5 abundance was quantified ( right panel ) .

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

(I) IB analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/ml CHX for the indicated time period before harvesting (left panel). The SIRT5 protein abundance was quantified (right panel).


( J ) IB of WCLs derived from TRIM21 + / + TRIM21 - / - BMDMs treated with 100 ng / mL LPS for indicated time period before harvesting ( top panel ) . SIRT5 abundance was quantified ( bottom panel ) .

(J) IB analysis of WCLs derived from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for the indicated time period before harvesting (top panel). The SIRT5 protein abundance was quantified (bottom panel).


( A ) Immunoblot of WCLs anti - GST immunoprecipitates derived from 293T transfected with indicated plasmids for 48 hours , were then treated with proteasome inhibitor MG132 for another 4 hours prior to harvesting .

O O O O O O O O O O O O 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) Immunoblot analysis of WCLs and anti-GST immunoprecipitates derived from 293T cells transfected with the indicated plasmids for 48 hours, cells were then treated with the proteasome inhibitor MG132 for another 4 hours prior to harvesting.


( B ) WCLs from TRIM21 + / + TRIM21 - / - BMDMs treated with 100 ng / mL LPS for 4 hours were immunoprecipitated with anti - SIRT5 antibody analyzed by western blotting . 10 μM MG132 was added for 4 hours before harvesting . Data information : IP WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(B) WCLs from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for 4 hours were immunoprecipitated with anti-SIRT5 antibody and analyzed by western blotting. 10 μM MG132 was added for 4 hours before harvesting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( C ) TRIM21 ubiquitinates SIRT5 in vitro . Immunopurified SIRT5 were incubated with purified recombinant TRIM21 or mutant TRIM21 , E1 E2 ( UBE2D2 ) , ubiquitin as indicated in 30 ℃ for 90 min . ubiquitination reaction products were resolved by SDS - PAGE probed with indicated antibodies .

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

(C) TRIM21 ubiquitinates SIRT5 in vitro. Immunopurified SIRT5 were incubated with purified recombinant TRIM21 or mutant TRIM21 proteins, E1 and E2 (UBE2D2), and ubiquitin as indicated in 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( E ) Purified SIRT5 mutant SIRT5 - 10KR were incubated with purified E1 E2 ( UBE2D2 ) , ubiquitin / or purified recombinant TRIM21 or control mIgG or GST as indicated at 30 ℃ for 90 min . SIRT5 ubiquitination TRIM21 were analyzed by western blotting with indicated antibodies . Data information : WB data in this work otherwise indicated are representative of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Purified SIRT5 and mutant SIRT5-10KR proteins were incubated with purified E1 and E2 (UBE2D2), ubiquitin and/or purified recombinant TRIM21 or control mIgG or GST proteins as indicated at 30℃ for 90 min. SIRT5 ubiquitination and TRIM21 protein levels were analyzed by western blotting with the indicated antibodies. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( A ) In vivo ubiquitination of WCLs GST - tagged immunoprecipitated products derived from 293T transfected with indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

O O O O O O O O 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) In vivo ubiquitination assay analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( B ) IB of WCLs anti - HA immunoprecipitates of 293T transfected with indicated plasmids . 48 hours posttransfection , were treated with 10 μM MG132 for 4 hours before harvesting .

O O O O O O O O O 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) IB analysis of WCLs and anti-HA immunoprecipitates of 293T cells transfected with the indicated plasmids. 48 hours posttransfection, cells were treated with 10 μM MG132 for 4 hours before harvesting.


( C ) Immunopurified SIRT5 were incubated with purified recombinant E1 ubiquitin ( Ub ) or mutant Ub - K48 or Ub - K63 in presence or absence of purified E2 ( UBE2D2 ) / or TRIM21 as indicated in 30 ℃ for 90 min . ubiquitination reaction products were resolved by SDS - PAGE probed with indicated antibodies .

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

(C) Immunopurified SIRT5 proteins were incubated with purified recombinant E1 and ubiquitin (Ub) or mutant Ub-K48 or Ub-K63 in the presence or absence of purified E2 (UBE2D2) and/or TRIM21 proteins as indicated in 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( D ) Immunopurified SIRT5 were incubated with purified recombinant TRIM21 , E1 E2 ( UBE2D2 ) , ubiquitin ( Ub ) or mutant Ub - K48R or Ub - K63R as indicated for 45 min at 30 ℃ . ubiquitination reaction products were resolved by SDS - PAGE probed with indicated antibodies .

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

(D) Immunopurified SIRT5 proteins were incubated with purified recombinant TRIM21, E1 and E2 (UBE2D2), and ubiquitin (Ub) or mutant Ub-K48R or Ub-K63R proteins as indicated for 45 min at 30℃. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( E ) WCLs from TRIM21 + / + TRIM21 - / - BMDMs treated with 100 ng / mL LPS for 4 hours were immunoprecipitated with anti - SIRT5 antibody analyzed by western blotting using antibodies specifically against Ub - K48 Ub - K63 . 10 μM MG132 was added for 4 hours before harvesting . Data information : IP WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(E) WCLs from TRIM21+/+ and TRIM21-/- BMDMs treated with 100 ng/mL LPS for 4 hours were immunoprecipitated with anti-SIRT5 antibody and analyzed by western blotting using antibodies specifically against Ub-K48 and Ub-K63. 10 μM MG132 was added for 4 hours before harvesting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( A ) Lysates from 293T transfected with HA - HAUSP Flag - SIRT5 or vector control ( - ) as indicated were immunoprecipitated ( IP ) using anti - Flag antibody , bound were analyzed by western blotting ( WB ) . Data information : IP WB data in this work otherwise indicated are representative of at least three independent experiments .

(A) Lysates from 293T cells transfected with HA-HAUSP and Flag-SIRT5 or vector control (-) as indicated were immunoprecipitated (IP) using anti-Flag antibody, and bound proteins were analyzed by western blotting (WB). Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( B , C ) Lysates from BMDMs were immunoprecipitated with anti - HAUSP ( B ) , anti - SIRT5 ( C ) or control IgG antibodies as indicated then analyzed by western blotting . Data information : IP WB data in this work otherwise indicated are representative of at least three independent experiments .

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

(B, C) Lysates from BMDMs were immunoprecipitated with anti-HAUSP (B), anti-SIRT5 (C) or control IgG antibodies as indicated and then analyzed by western blotting. Data information: All IP and WB data in this work otherwise indicated are representative of at least three independent experiments.


( D ) IB of WCLs anti - SIRT5 immunoprecipitates of BMDMs treated with 100 ng / ml LPS for 4 hours . were treated with 10 μM MG132 for 4 hours before harvesting .

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

(D) IB analysis of WCLs and anti-SIRT5 immunoprecipitates of BMDMs treated with 100 ng/ml LPS for 4 hours. Cells were treated with 10 μM MG132 for 4 hours before harvesting.


( E ) 293T were transfected with Flag - tagged HAUSP , mutant HAUSP - CS ( C223S ) or vector control as indicated for 48 hours . expression was determined by western blot using indicated antibodies . Data information : WB data in this work otherwise indicated are representative of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were transfected with Flag-tagged HAUSP, mutant HAUSP-CS (C223S) or vector control as indicated for 48 hours. protein expression was determined by western blot analysis using indicated antibodies. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( F ) IB of WCLs derived from 293T transfected with HA - SIRT5 together with Flag - HAUSP or vector control constructs ( left panel ) . were treated with 100 ng / ml CHX for indicated time period prior to harvesting . SIRT5 abundance was quantified ( right panel ) .

O O O O O O O O O O O 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

(F) IB analysis of WCLs derived from 293T cells transfected with HA-SIRT5 together with Flag-HAUSP or vector control constructs (left panel). Cells were treated with 100 ng/ml CHX for the indicated time period prior to harvesting. The SIRT5 protein abundance was quantified (right panel).


( G , H ) WCLs from BMDMs treated with increasing amounts of P5091 ( G ) or P22077 ( H ) in presence or absence of 100 ng / mL LPS were subjected to IB using indicated antibodies ( left panel ) . SIRT5 abundance was quantified ( right panel ) .

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 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

(G, H) WCLs from BMDMs treated with increasing amounts of P5091(G) or P22077 (H) in the presence or absence of 100 ng/mL LPS were subjected to IB analysis using indicated antibodies (left panel). The SIRT5 protein abundance was quantified (right panel).


( B ) IB of WCLs anti - GST immunoprecipitates of 293T transfected with indicated plasmids in presence or absence of 100 µM P22077 for 24 hours . were treated with 10 μM MG132 for 4 hours before harvesting .

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

(B) IB analysis of WCLs and anti-GST immunoprecipitates of 293T cells transfected with the indicated plasmids in the presence or absence of 100 µM P22077 for 24 hours. Cells were treated with 10 μM MG132 for 4 hours before harvesting.


( C , IB of WCLs GST - tagged immunoprecipitated products derived from 293T transfected with indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

O O O O O O 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, IB analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


D ) IB of WCLs GST - tagged immunoprecipitated products derived from 293T transfected with indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

O O O O O 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) IB analysis of WCLs and GST-tagged immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( E ) In vitro SIRT5 deubiquitination . Ubiquitinated SIRT5 was pulled down from 293T cotransfected HA - Ub , then incubated with purified wild - type HAUSP or catalytically inactive HAUSP mutant ( HAUSP - CS ) 30 ℃ for 90 min . reaction products were resolved by SDS - PAGE probed with indicated antibodies .

(E) In vitro SIRT5 deubiquitination assay. Ubiquitinated SIRT5 was pulled down from 293T cells cotransfected HA-Ub, and then incubated with purified wild-type HAUSP or the catalytically inactive HAUSP mutant (HAUSP-CS) 30℃ for 90 min. The reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( F ) IB of WCLs anti - SIRT5 immunoprecipitated products derived from BMDMs transfected with control or HAUSP siRNA for 48 hours . 10 μM MG132 was added for 6 hours before harvesting .

O O O O O O O O O O O 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) IB analysis of WCLs and anti-SIRT5 immunoprecipitated products derived from BMDMs transfected with control or HAUSP siRNA for 48 hours. 10 μM MG132 was added for 6 hours before harvesting.


( G ) BMDMs transfected with control ( - ) or HAUSP siRNA for 48 hours were then treated with 100 ng / mL mM LPS for 4 hours , 10 μM MG132 was added for 4 hours before harvesting . WCLs anti - SIRT5 immunoprecipitated products were prepared subjected to IB using indicated antibodies .

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

(G) BMDMs transfected with control (-) or HAUSP siRNA for 48 hours were then treated with 100 ng/mL mM LPS for 4 hours, and 10 μM MG132 was added for 4 hours before harvesting. WCLs and anti-SIRT5 immunoprecipitated products were prepared and subjected to IB analysis using indicated antibodies.


( A ) IB of WCLs anti - Flag immunoprecipitated products derived from 293T transfected with indicated plasmids using anti - Flag , anti - Acetyllysine , anti - Succinyllysine , anti - Glutaryllysine anti - Malonyllysine

O 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 B-MEASURED_VAR

(A) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids using anti-Flag, anti-Acetyllysine, anti-Succinyllysine, anti-Glutaryllysine and anti-Malonyllysine antibodies


( B ) IB of WCLs anti - TRIM21 immunoprecipitated products derived from 293T transfected with control or SIRT5 siRNA using anti - Flag , anti - acetyl Lysine ( AcK ) , anti - succinyl Lysine ( SulK ) , anti - glutaryl Lysine ( GluK ) anti - malonyl Lysine ( MalK ) antibodies .

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

(B) IB analysis of WCLs and anti-TRIM21 immunoprecipitated products derived from 293T cells transfected with control or SIRT5 siRNA using anti-Flag, anti-acetyl Lysine (AcK), anti-succinyl Lysine (SulK), anti-glutaryl Lysine (GluK) and anti-malonyl Lysine (MalK) antibodies.


( C ) IB of WCLs anti - Flag immunoprecipitated products derived from 293T transfected with indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

(C) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( D ) IB of WCLs anti - TRIM21 immunoprecipitated products derived from BMDMs treated 100 ng / mL LPS or left untreated for 4 hours . 10 μM MG132 was added for 4 hours before harvesting .

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

(D) IB analysis of WCLs and anti-TRIM21 immunoprecipitated products derived from BMDMs treated 100 ng/mL LPS or left untreated for 4 hours. 10 μM MG132 was added for 4 hours before harvesting.


( E ) expression in SIRT5 + / + SIRT5 - / - BMDMs was determined by western blot . Data information : WB data in this work otherwise indicated are representative of at least three independent experiments .

O O O O O O O O O O O O O O O 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) Protein expression in SIRT5+/+ and SIRT5-/- BMDMs was determined by western blot analysis. Data information: All WB data in this work otherwise indicated are representative of at least three independent experiments.


( F , IB of WCLs anti - Flag immunoprecipitated products derived from 293T transfected with indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

(F, IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


G ) IB of WCLs anti - Flag immunoprecipitated products derived from 293T transfected with indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

G) IB analysis of WCLs and anti-Flag immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( H ) IB of WCLs anti - GST immunoprecipitated products derived from 293T transfected with indicated plasmids in presence or absence of 3 mM NAM for 24 hours . 10 μM MG132 was added for 4 hours before harvesting .

O 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

(H) IB analysis of WCLs and anti-GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids in the presence or absence of 3 mM NAM for 24 hours. 10 μM MG132 was added for 4 hours before harvesting.


( I ) Purified TRIM21 , TRIM21 - K351R TRIM21 - K385R were incubated with purified E1 E2 ( UBE2D2 ) , ubiquitin SIRT5 as indicated at 30 ℃ for 90 min . ubiquitination reaction products were resolved by SDS - PAGE probed with indicated antibodies .

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

(I) Purified TRIM21, TRIM21-K351R and TRIM21-K385R proteins were incubated with purified E1 and E2 (UBE2D2), ubiquitin and SIRT5 proteins as indicated at 30℃ for 90 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the indicated antibodies.


( J ) IB of WCLs anti - GST immunoprecipitated products derived from 293T transfected with indicated plasmids . 10 μM MG132 was added for 4 hours before harvesting .

(J) IB analysis of WCLs and anti-GST immunoprecipitated products derived from 293T cells transfected with the indicated plasmids. 10 μM MG132 was added for 4 hours before harvesting.


( E ) Genotyping of TRIM21 - / - , SIRT5 - / - TRIM21 - / - SIRT5 - / - mice . derived from tail of wild type knockout was analyzed by PCR . expression of TRIM21 SIRT5 in BMDMs derived from mice were also analyzed by western blotting . Data information : Western blots in E represent three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genotyping of TRIM21-/-, SIRT5-/- and TRIM21-/-SIRT5-/- mice. DNA derived from the tail of wild type and knockout animals was analyzed by PCR. Protein expression of TRIM21 and SIRT5 in BMDMs derived from mice were also analyzed by western blotting. Data information: Western blots in E represent three independent experiments.


wild - type ( WT , n = 5 ) , TRIM21 - / - ( n = 5 ) , SIRT5 - / - ( n = 5 ) , TRIM21 - / - SIRT5 - / - ( n = 5 ) mice were orally administrated with 2 . 5 % ( w / v ) DSS in drinking water for 7 days regular drinking water for another 2 days . Colon - ( G ) changes were measured . Data information : Data are means ± SD . n = 5 biological replicates . P values were determined by unpaired two - Student ' s t - . ns , not significant ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

wild-type (WT, n=5), TRIM21-/- (n=5), SIRT5-/-(n=5), and TRIM21-/-SIRT5-/- (n=5) mice were orally administrated with 2.5% (w/v) DSS in drinking water for 7 days and regular drinking water for another 2 days. Colon-length (G) changes were measured. Data information: Data are means ± SD. n=5 biological replicates. P values were determined by unpaired two-tailed Student's t-tests. ns, not significant; ***p < 0.001, ****p < 0.0001.


( C ) Expression profile of indicated ion channel analyzed by RT - PCR in WM266 . 4 451Lu metastatic melanoma lines ( middle bottom panels , respectively ) . Expected amplicons sizes ( positive controls in top panel ) are in base pairs ( bp ) ; TRPV1 = 120 , TRPV2 = 199 , TRPV3 = 226 , TRPV4 = 190 / 370 , TRPV6 = 208 , TRPC1 = 201 , TRPC6 = 121 , TRPM2 = 303 , TRPM7 = 226 , TRPA1 = 140 , Orai1 = 161 , STIM1 = 109 , STIM2 = 114 ) .

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(C) Expression profile of the indicated ion channel analyzed by RT-PCR in the WM266.4 and 451Lu metastatic melanoma cell lines (middle and bottom panels, respectively). Expected amplicons sizes (positive controls in top panel) are in base pairs (bp); TRPV1=120, TRPV2=199, TRPV3=226, TRPV4=190/370, TRPV6=208, TRPC1=201, TRPC6=121, TRPM2=303, TRPM7=226, TRPA1=140, Orai1=161, STIM1=109, STIM2=114).


( F ) TRPV2 immunoblotting in 36 out of 60 NCI - 60 lines . Tubulin was used as a loading control . Note that after longer exposure times , TRPV2 expression was also detected in some breast prostate cancer lines , as it has been previously described ( Elbaz et al , 2018 ; Gambade et al , 2016 ; Monet et al , 2010 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TRPV2 immunoblotting in 36 out of the 60 NCI-60 cell lines. Tubulin was used as a loading control. Note that after longer exposure times, TRPV2 expression was also detected in some breast and prostate cancer cell lines, as it has been previously described (Elbaz et al, 2018; Gambade et al, 2016; Monet et al, 2010).


( G ) TRPV2 expression in melanoma lines harboring either B - RAF ( black ) or N - Ras ( blue ) mutations . β - Actin was used as a loading control .

(G) TRPV2 protein expression in melanoma cell lines harboring either B-RAF (black) or N-Ras (blue) mutations. β-Actin was used as a loading control.


( A ) for total plasma ( PM ) TRPV2 expression in melanoma lines . β - actin was used as loading integrity control . PM fractions were isolated using surface biotinylation technique . higher molecular bands of TRPV2 correspond to glycosylated mature

O O O O O O O O O O O O O O O O O O O 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) Analysis for total and plasma membrane (PM) TRPV2 expression in melanoma cell lines. β-actin was used as loading and cell integrity control. PM fractions were isolated using the surface protein biotinylation technique. The higher molecular weight bands of TRPV2 correspond to the glycosylated and mature channel


( C ) TRPV2 overexpression in non - invasive 501mel ( GFP - TRPV2 ) or downregulation by lentiviral - delivery of TRPV2 specific shRNAs ( shRNA V2 - 1 V2 - 2 ) in WM266 . 4 451Lu metastatic melanoma lines assessed by western - blot . β - actin was used as a loading control .

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(C) TRPV2 overexpression in the non-invasive 501mel cell line (GFP-TRPV2) or downregulation by lentiviral-delivery of TRPV2 specific shRNAs (shRNA V2-1 and V2-2) in the WM266.4 and 451Lu metastatic melanoma cell lines assessed by western-blot. β-actin was used as a loading control.


( E ) Average normalized quenching rates ( as in D ) presented as mean ± ( n > 3 biological replicates ) . * * P = 0 . 0095 , Mann - Whitney ( left panel ) ; * P = 0 . 0399 - 0 . 0336 ( middle panel ) * P = 0 . 0438 , * * P = 0 . 0033 ( right panel ) Kruskal - Wallis followed by Dunn ' s multiple comparisons .

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(E) Average normalized quenching rates (as in D) presented as mean ± SEM (n>3 biological replicates). **P = 0.0095, the Mann-Whitney test (left panel); *P = 0.0399-0.0336 (middle panel) and *P = 0.0438, **P = 0.0033 (right panel) the Kruskal-Wallis test followed by Dunn's multiple comparisons test.


( A ) Impact of TRPV2 genetic manipulation in 501mel , WM266 . 4 451Lu on serum - induced migration matrigel invasion . Representative pictures show crystal violet - stained that have migrated through 8 µm pore membranes . Histograms illustrate average numbers of migrating / invading normalized to control ( presented as mean ± of n = 3 independent experiments ) . For 501mel Mann - Whitney was used for statistical ( * * P = 0 . 0022 ) ; For WM266 . 4 & 451Lu one - way ANOVA followed by Dunnett ' s multiple comparisons was used ( * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; See appendix Table S2 for exact P values ) .

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(A) Impact of TRPV2 genetic manipulation in 501mel, WM266.4 and 451Lu on serum-induced migration and matrigel invasion. Representative pictures show crystal violet-stained cells that have migrated through 8 µm pore size membranes. Histograms illustrate the average numbers of migrating/invading cells normalized to control (presented as mean ± SEM of n=3 independent experiments). For 501mel the Mann-Whitney test was used for statistical analysis (**P = 0.0022); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; ***P < 0.001; ****P < 0.0001 ; See appendix Table S2 for exact P values).


( D ) TRPV2 - silencing effect on 3D invasion . Representative were taken every day for 3 days after collagen embedding of spheroids from WM266 . 4 expressing either shRNA ctrl or TRPV2 - targeting shRNAs ( scale bar = 0 . 5 mm ) . ( E ) of collagen invasion . For each spheroid , - covered at day 2 was normalized to starting of collagen - embedded spheroids . Histograms represent invasion relative to control WM266 . 4 spheroids ( shRNA Ctrl ) from at least 12 spheroids from three independent experiments ( See also appendix Fig S3C ) . M . * * * * P < 0 . 0001 * * P = 0 . 0012 Kruskal - Wallis followed by Dunn ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TRPV2-silencing effect on 3D invasion. Representative images were taken every day for 3 days after collagen embedding of spheroids from WM266.4 cells expressing either shRNA ctrl or TRPV2-targeting shRNAs (scale bar=0.5 mm). (E) Quantification of collagen invasion. For each spheroid, the cell-covered area at day 2 was normalized to the starting area of the collagen-embedded spheroids. Histograms represent the invasion relative to control WM266.4 spheroids (shRNA Ctrl) from at least 12 spheroids from three independent experiments (See also appendix Fig S3C). M. ****P <0.0001 and **P = 0.0012 the Kruskal-Wallis test followed by Dunn's multiple comparisons test.


( B ) Representative confocal of in situ detection of endogenous TRPV2 interactions with paxillin vinculin by proximity - ligation assays ( PLA ) . Low confluency WM266 . 4 451Lu metastatic melanoma plated on fibronectin - coated coverslips were stained for F - actin ( green ) , nuclei ( blue ) PLA reaction using antibodies specific of indicated ( red ) ( scale bar = 20 µm ; identical for pictures ) . Red spots indicate association of two of interest , close to 40 nm . Scatter plots represent of number of PLA spots per ( bars indicate medians ) between TRPV2 a control antibody ( IgG ) , paxillin ( PAX ) or vinculin ( VCL ) . ( n = 29 - 51 from at least three independent experiments ) .

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(B) Representative confocal images of the in situ detection of endogenous TRPV2 interactions with paxillin and vinculin by proximity-ligation assays (PLA). Low confluency WM266.4 and 451Lu metastatic melanoma cells plated on fibronectin-coated coverslips were stained for F-actin (green), cell nuclei (blue) and PLA reaction using antibodies specific of the indicated proteins (red) (scale bar = 20 µm ; identical for all pictures). Red fluorescent spots indicate the association of the two proteins of interest, close to 40 nm. Scatter plots represent the quantification of the number of PLA spots per cell (bars indicate the medians) between TRPV2 and a control antibody (IgG), paxillin (PAX) or vinculin (VCL). (n=29-51 cells from at least three independent experiments).


( C ) Super - resolution ( dSTORM ) imaging of clustering of TRPV2 channels with paxillin , but not with vinculin , in migrating WM266 . 4 ( Scale bar = 5µm ) . a b insets show expanded views of a region of arrows highlight TRPV2 channel paxillin co - clusters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Super-resolution (dSTORM) imaging of the clustering of TRPV2 channels with paxillin, but not with vinculin, in migrating WM266.4 cells (Scale bar = 5µm). a and b insets show expanded views of a region of the cell and arrows highlight TRPV2 channel and paxillin co-clusters.


( B ) Comparison of calpain activity in control ( GFP ) or overexpressing TRPV2 ( GFP - TRPV2 ) 501mel , in control ( shRNA Ctrl ) or TRPV2 - silenced ( shRNA V2 - 1 , - 2 ) WM266 . 4 or 451Lu . Bar graphs show mean normalized calpain activity ± ( n = 3 biological replicates ) . For 501mel Mann - Whitney was used for statistical ( * P = 0 . 05 ) ; For WM266 . 4 & 451Lu one - way ANOVA followed by Dunnett ' s multiple comparisons was used ( * * P < 0 . 01 ; See appendix Table S2 for exact P values ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of calpain activity in control (GFP) or overexpressing TRPV2 (GFP-TRPV2) 501mel cells, and in control (shRNA Ctrl) or TRPV2-silenced (shRNA V2-1, -2) WM266.4 cells or 451Lu cells. Bar graphs show mean normalized calpain activity ± SEM (n=3 biological replicates). For 501mel the Mann-Whitney test was used for statistical analysis (*P = 0.05); For WM266.4&451Lu one-way ANOVA followed by Dunnett's multiple comparisons tests was used (**P < 0.01; See appendix Table S2 for exact P values).


( C ) Representative immunoblots showing full - ( 230 kDa ; Black Arrows ) calpain - mediated degradation product ( 190 kDa ; Grey Arrows ) of talin after overexpression or repression of TRPV2 , in corresponding lines . Tubulin was used as a loading control .

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(C) Representative immunoblots showing the full-length (230 kDa ; Black Arrows) and the calpain-mediated degradation product (190 kDa ; Grey Arrows) of talin after overexpression or repression of TRPV2, in the corresponding cell lines. Tubulin was used as a loading control.


A - D Representative bioluminescence imaging ( BLI ) data of mice injected intravenously with non - metastatic melanoma 501mel - Luc transfected with either GFP - TRPV2 or GFP control ( A - B ) , or with invasive 451Lu - Luc melanoma expressing either control shRNA or TRPV2 targeting shRNA ( C - D ) . growth metastasis formation were monitored for 64 days ( 501mel - Luc ) or 35 days ( 451Lu - Luc ) after injection . Graphs presented in A C show in vivo normalized photon flux at end time point ( ns P = 0 . 0903 ( A ) * * P = 0 . 0064 ( C ) , Mann - Withney ) . Graphs presented in B D show number of metastatic foci per ( B ) or per lungs ( D ) counted at necropsy ( * P = 0 . 0397 ( B ) ns P = 0 . 0691 ( D ) , unpaired t - ) . For ( D ) Representative ex vivo BLI of lung metastasis are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Representative bioluminescence imaging (BLI) data of mice injected intravenously with the non-metastatic melanoma cell line 501mel-Luc transfected with either GFP-TRPV2 or GFP control (A-B), or with the invasive 451Lu-Luc melanoma cell line expressing either control shRNA or TRPV2 targeting shRNA (C-D). Tumor growth and metastasis formation were monitored for 64 days (501mel-Luc) or 35 days (451Lu-Luc) after injection. Graphs presented in A and C show in vivo normalized photon flux quantification at the end time point (ns P = 0.0903 (A) and **P = 0.0064 (C), the Mann-Withney test). Graphs presented in B and D show the number of metastatic foci per animal (B) or per lungs (D) counted at necropsy (*P = 0.0397 (B) and ns P = 0.0691 (D), unpaired t-tests). For (D) Representative ex vivo BLI images of lung metastasis are shown


( A ) TRPV2 expression ( microarray probe : 219282 _ s _ at ) measured in normal skin ( n = 7 biological replicates ) , nevi ( n = 9 biological replicates ) , ( n = 31 biological replicates ) metastatic ( n = 73 biological replicates ) melanomas ( GEO : GSE46517 ) ( Data ref : Kwong LN , 2013 ) . TRPV2 are increased in malignant lesions compared to normal skin nevi . Data is represented as a whisker - box plot with outliers plotted as points ( Boxes extend from 25th to 75th percentiles , whiskers from 10th to 90th percentiles , horizontal in each box is plotted at median ; ns P = 0 , 1383 , * P = 0 , 0488 * * P = 0 , 0016 , Kruskal - Wallis ) . horizontal dotted corresponds to TRPV2 median expression in nevi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TRPV2 RNA expression levels (microarray probe: 219282_s_at) measured in normal skin (n=7 biological replicates), nevi (n=9 biological replicates) , primary (n=31 biological replicates) and metastatic (n=73 biological replicates) melanomas (GEO: GSE46517)(Data ref: Kwong LN, 2013). TRPV2 levels are increased in malignant lesions compared to normal skin and nevi. Data is represented as a whisker-box plot with outliers plotted as individual points (Boxes extend from the 25th to 75th percentiles, whiskers from the 10th to 90th percentiles, the horizontal line in each box is plotted at the median; ns P = 0,1383, *P = 0,0488 and **P = 0,0016, the Kruskal-Wallis test). The horizontal dotted line corresponds to TRPV2 median expression in nevi.


( B ) Representative melanoma tissue lesions from a tissue microarray ( TMA ) comparing TRPV2 between a benign nevus a malignant melanoma ( See also appendix Fig S10 ) ( Scale bar = 200µm ) . Top right insets show same stained with an anti - melanoma triple cocktail ( HMB45 + A103 + T311 ) . Regions depicted with a black represent interface between diffusely stained ( T ) surrounding normal stroma ( S ) . Right panel shows scatter plot of complete of TRPV2 in 77 with malignant melanoma compared to 16 nevi tissues ( dot , single sample ) with lines for mean ± ( * P = 0 . 0473 , Mann - Whitney ) .

(B) Representative melanoma tissue lesions from a tissue microarray (TMA) comparing TRPV2 staining between a benign nevus and a malignant melanoma (See also appendix Fig S10) (Scale bar = 200µm). Top right insets show the same images stained with an anti-melanoma triple cocktail (HMB45+A103+T311). Regions depicted with a black line represent the interface between the diffusely stained tumor (T) and the surrounding normal stroma (S). Right panel shows the scatter plot of the complete quantification of TRPV2 staining in 77 patients with malignant melanoma compared to 16 nevi tissues (dot, single patient sample) with lines for mean ± SEM (*P = 0.0473, the Mann-Whitney test).


( F ) of TRPV2 expression by quantitative RT - PCR ( left panel ) western - blot ( right panel ) in WM115 / WM266 . 4 pair of isogenic melanoma lines . WM115 was derived from an in situ while WM266 . 4 was established from a skin metastasis of same . Relative transcript are presented as mean ± from n = 4 - 9 biological replicates ( * * P = 0 . 0028 , Mann - Whitney ) .

(F) Analysis of TRPV2 expression by quantitative RT-PCR (left panel) and western-blot (right panel) in the WM115/WM266.4 pair of isogenic melanoma cell lines. The WM115 cell line was derived from an in situ tumor while the WM266.4 was established from a skin metastasis of the same patient. Relative transcript levels are presented as mean ± SEM from n=4-9 biological replicates (**P = 0.0028, the Mann-Whitney test).


( G ) Kaplan - Meier plot showing association of TRPV2 expression ( 10 % highest versus 10 % lowest expressers in skin cutaneous melanoma cohort TCGA data set ) with melanoma survival ( ( * ) P = 0 , 0144 , Log - rank ( Mantel - Cox ) ) .

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(G) Kaplan-Meier plot showing the association of TRPV2 expression (10% highest versus 10% lowest expressers in the skin cutaneous melanoma cohort TCGA data set) with melanoma patient survival ((*)P = 0,0144, the Log-rank (Mantel-Cox) test).


1 . SARS - CoV - 2 activates delayed innate immune responses in lung epithelial Measurements of replication innate immune induction in Calu - 3 lung epithelial infected with SARS - CoV - 2 at MOIs 0 . 08 , 0 . 4 2 TCID50VERO / . ( A ) Replication of SARS - CoV - 2 genomic subgenomic E RNAs ( qRT - PCR ) . ( B ) of N from in ( A ) by flow cytometry . Mean of N - positive of live - gated is shown + / - , n = 2 .

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1.SARS-CoV-2 activates delayed innate immune responses in lung epithelial cells Measurements of replication and innate immune induction in Calu-3 lung epithelial cells infected with SARS-CoV-2 at MOIs 0.08, 0.4 and 2 TCID50VERO/cell. (A) Replication of SARS-CoV-2 genomic and subgenomic E RNAs (qRT-PCR). (B) Quantification of N staining from cells in (A) by flow cytometry. Mean percentage of N-positive of all live-gated cells is shown +/- SEM, n=2.


( C ) Representative example of immunofluorescence of N ( green ) after SARS - CoV - 2 infection of Calu - 3 at MOI 0 . 4 TCID50VERO / , at time points shown . Nuclei ( DAPI , blue ) , mask ( red ) . Scale bar represents 50µM .

(C) Representative example of immunofluorescence staining of N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue), cell mask (red). Scale bar represents 50µM.


Fold induction of ( F ) interferons ( IFNβ , IFNλ1 IFNλ3 ) pro - inflammatory mediators ( IL - 6 CCL5 ) each overlaid with SARS - CoV - 2 E ( qRT - PCR ) .

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Fold induction of (F) interferons (IFNβ, IFNλ1 and IFNλ3) pro-inflammatory mediators (IL-6 and CCL5) each overlaid with SARS-CoV-2 E (qRT-PCR).


Fold induction of IFN stimulated genes ( CXCL10 IFIT2 ) each overlaid with SARS - CoV - 2 E ( qRT - PCR ) .

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Fold induction of IFN stimulated genes (CXCL10 and IFIT2) each overlaid with SARS-CoV-2 E (qRT-PCR).


Peak SARS - CoV - 2 replication precedes innate immune activation . ( A - I ) ( A , C ) Representative of NF - κB p65 ( A ) ( red ) IRF3 ( C ) ( red ) localisation in mock or SARS - CoV - 2 infected ( MOI 0 . 4 TCID50VERO / ) Calu - 3 at 24 hpi . SARS - CoV - 2 N ( green . ( E G ) Representative of IL - 6 mRNA ( E ) detected by FISH ( red ) N ( green ) , or IFIT1 mRNA ( G ) ( green ) with N ( red ) , both with nuclei ( DAPI , blue ) in mock or SARS - CoV - 2 infected ( MOI 0 . 4 TCID50VERO / ) Calu - 3 at 24 hpi . ( B , D , F , H , I ) Single time course quantifying Integrated of NF - κB p65 ( B ) , IRF3 ( D ) , or overall integrated for IL - 6 ( F ) or IFIT1 ( H ) mRNA over time in N positive N negative ( I ) . n = 2 . Kruskal - Wallis with Dunn ' s multiple comparison . * ( p < 0 . 05 ) , * * * * ( p < 0 . 0001 ) . Scale bar represents 50µM .

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Peak SARS-CoV-2 replication precedes innate immune activation. (A-I) (A,C) Representative images of NF-κB p65 (A) (red) and IRF3 (C) (red) nuclear localisation in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. SARS-CoV-2 N protein (green. (E and G) Representative images of IL-6 mRNA (E) detected by FISH (red) and N protein (green) , or IFIT1 mRNA (G) (green) with N protein (red), both with nuclei (DAPI, blue) in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. (B, D, F, H, I) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF-κB p65 (B), IRF3 (D) , or overall integrated intensity for IL-6 (F) or IFIT1 (H) mRNA over time in N protein positive cells and N protein negative cells (I). n=2. Kruskal-Wallis test with Dunn's multiple comparison. * (p<0.05), **** (p<0.0001). Scale bar represents 50µM.


( E - H ) Measurement of ( E ) viral genomic subgenomic E ( F ) fold induction of CXCL10 , ( G ) IFIT2 ( H ) IL - 6 at 24 hpi , of Calu - 3 with SARS - CoV - 2 ( MOI 0 . 04 TCID50VERO / ) with Remdesivir treatment ( 5μM ) prior to , at time of , or 8 h post - infection . Mean + / - , n = 3 , One way ANOVA with Dunnett ' s multiple comparisons to compare to untreated infected condition ( ' mock ' ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) , * * * * ( p < 0 . 0001 ) .

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(E-H) Measurement of (E) viral genomic and subgenomic E RNA (F) fold induction of CXCL10, (G) IFIT2 and (H) and IL-6 at 24 hpi, of Calu-3 cells with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) with Remdesivir treatment (5μM) prior to, at the time of, or 8 h post-infection. Mean +/- SEM, n=3, One way ANOVA with Dunnett's multiple comparisons test to compare to untreated infected condition ('mock'), ** (p<0.01), *** (p<0.001), **** (p<0.0001).


( I ) Representative example of immunofluorescence of dsRNA ( red ) N ( green ) after SARS - CoV - 2 infection of Calu - 3 at MOI 0 . 4 TCID50VERO / , at time points shown . Nuclei ( DAPI , blue ) . Scale bar represents 50µM .

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(I) Representative example of immunofluorescence staining of dsRNA (red) and N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue). Scale bar represents 50µM.


( Q - R ) ( Q ) Viral E ( R ) released infectious ( TCID50VERO / ) at 24 hpi of infected Calu - 3 depleted for MAVs , RIG - I or MDA5 . Mean + / - , n = 3 . Each group compared to siCtrl by One Way ANOVA with Dunnett ' s multiple comparisons , * , p > 0 . 05 , * * ( p < 0 . 01 ) , n . s : non significant .

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(Q-R) (Q) Viral E RNA and (R) released infectious virus (TCID50VERO/cell) at 24 hpi of infected Calu-3 cells depleted for MAVs, RIG-I or MDA5. Mean +/- SEM, n=3. Each group compared to siCtrl by One Way ANOVA with Dunnett's multiple comparisons test, *, p>0.05, ** (p<0.01), n.s : non significant.


( G - H ) Viral genomic subgenomic E at 24 hpi ( RT - qPCR ) with DMSO or TPCA ( G ) or Rux ( H ) treatment . Data information : ( A - H ) Mean + / - , n = 3 , statistical comparisons are made by unpaired t comparing inhibitor - treated to mock - treated SARS - CoV - 2 infected conditions at each MOI each timepoint . * ( p < 0 . 05 ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) , * * * * ( p < 0 . 0001 ) .

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(G-H) Viral genomic and subgenomic E RNA at 24 hpi (RT-qPCR) with DMSO or TPCA (G) or Rux (H) treatment. Data information: (A-H) Mean +/- SEM, n=3, statistical comparisons are made by unpaired t test comparing inhibitor-treated to mock-treated SARS-CoV-2 infected conditions at each MOI and each timepoint. * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001).


( B - J ) Calu - 3 were transfected with siRNA targeting MAVS or non - targeting control ( siCtrl ) ( B - D ) or treated with DMSO vehicle or inhibitors 10 μM TPCA - 1 ( E - G ) or 2 μM Ruxolitinib ( Rux ) ( H - J ) as shown , were mock - infected or infected with SARS - CoV - 2 at MOI 0 . 04 TCID50VERO / . containing conditioned media ( CoM ) was harvested at 48 hpi . MDM were treated with Calu - 3 containing CoM for 6 hpi , before washing measuring MDM gene expression ( B , E , H ) , MDM activation markers by flowcytometry 48 h later ( C , D , F , G , I , J ) , plotting relative median ( MFI ) compared to mock - infected siCtrl ( C , D ) or mock - infected DMSO control ( F , G , I , J ) . Legends in ( B ) , ( E ) ( H ) apply to ( C , D ) , ( F , G ) ( I , J ) respectively . inhibitors in ( E ) ( H ) were tested side - by - side with same mock condition . Mean + / - shown , data from 4 - 6 independent MDM donors is shown . Statistical comparison by two - paired t - comparing MDM exposed to control infected CoM to siMAVS / inhibitor treated infected CoM . * ( p < 0 . 05 ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) .

O O O O O O O O O O O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J) Calu-3 cells were transfected with siRNA targeting MAVS or non-targeting control (siCtrl) (B-D) or treated with DMSO vehicle or inhibitors 10 μM TPCA-1 (E-G) or 2 μM Ruxolitinib (Rux) (H-J) as shown, and were mock-infected or infected with SARS-CoV-2 at MOI 0.04 TCID50VERO/cell. Virus containing conditioned media (CoM) was harvested at 48 hpi. MDM were treated with Calu-3 virus containing CoM for 6 hpi, before washing and measuring MDM gene expression (B, E, H), and MDM activation markers by flowcytometry 48 h later (C,D,F,G,I,J) , plotting relative median fluorescent intensity (MFI) compared to mock-infected siCtrl (C, D) or mock-infected DMSO control (F, G, I, J). Legends in (B), (E) and (H) apply to (C,D), (F,G) and (I,J) respectively. The inhibitors in (E) and (H) were tested side-by-side with the same mock condition. Mean +/- SEM shown, data from 4-6 independent MDM donors is shown. Statistical comparison by two-tailed paired t-test comparing MDM exposed to control infected CoM to siMAVS/inhibitor treated infected CoM. * (p<0.05), ** (p<0.01), *** (p<0.001).


Calu - 3 were infected with SARS - CoV - 2 ( MOI 0 . 04 TCID50VERO / ) in presence or absence of 10ng / ml IL - 1b . ( U ) Non - adherent were collected at 48h post infection live / dead stained . were acquired by flowcytometry determined by time - gating . For statistical comparison , total numbers were compared . ( V ) Viral release into culture supernatants at 24 h was measured by TCID50 on VeroE6 . ( Q - V ) Mock SARS - CoV - 2 infected conditions were compared with or without IL1b - treatment , respectively , by unpaired T ( n = 3 ) . * , p < 0 . 05 ; n . s . , non - significant . Mean + / - shown .

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Calu-3 cells were infected with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) in the presence or absence of 10ng/ml IL-1b. (U) Non-adherent cells were collected at 48h post infection and live/dead stained. Cells were acquired by flowcytometry and cell counts determined by time-gating. For statistical comparison, total cell numbers were compared. (V) Viral release into culture supernatants at 24 h was measured by TCID50 on VeroE6 cells. (Q-V) Mock and SARS-CoV-2 infected conditions were compared with or without IL1b-treatment, respectively, by unpaired T test (n=3). *, p<0.05; n.s., non-significant. Mean +/- SEM shown.


A Immunoblot of overexpressed ALFA - tagged CNX in CNX knockout lines clones # 1 # 2 , which were used for mass spectrometry . Endogenous CNX level of wildtype HEK293T is shown as a control .

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A Immunoblot analysis of overexpressed and ALFA-tagged CNX in CNX knockout cell lines clones #1 and #2, which were used for mass spectrometry. Endogenous CNX level of wildtype HEK293T cells is shown as a control.


C Volcano plots derived from LC - MS / MS analyses of ALFA - tagged CNX , immunoprecipitated in 1 % NP - 40 buffer from transfected CNX deficient HEK293T after DSSO crosslinking compared to empty vector ( EV ) control co - IPs . Depicted is an enlarged section of associated complete volcano plot in upper right . Among significantly enriched , annotations are Uniprot gene names . CNX is shown in green . ER with GO - term annotation " endoplasmic reticulum " , which are localized to ER lumen , are highlighted in blue among those several known CNX interaction partners are indicated with their names . are shown in red those which are non - glycosylated are additionally labeled with their gene names .

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C Volcano plots derived from LC-MS/MS analyses of ALFA-tagged CNX, immunoprecipitated in 1% NP-40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector (EV) control co-IPs. Depicted is an enlarged section of the associated complete volcano plot in the upper right. Among the significantly enriched proteins, annotations are Uniprot gene names. CNX is shown in green. ER proteins with the GO-term annotation "endoplasmic reticulum", which are localized to the ER lumen, are highlighted in blue and among those several known CNX interaction partners are indicated with their names. Membrane proteins are shown in red and those which are non-glycosylated are additionally labeled with their gene names.


E Co - immunoprecipitations of endogenous CNX with FLAG - tagged Cx32 WT its mutants including statistical . Constructs were expressed in HEK293T . One representative immunoblot is shown . Monomers dimers of Cx32 are indicated with brackets . samples were normalized to

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E Co-immunoprecipitations of endogenous CNX with FLAG-tagged Cx32 WT and its mutants including quantification and statistical analysis. Constructs were expressed in HEK293T cells. One representative immunoblot is shown. Monomers and dimers of Cx32 are indicated with brackets. All samples were normalized to WT


F Immunofluorescence of Cx32 its mutants . COS - 7 were transiently transfected with indicated FLAG - tagged Cx32 constructs immunofluorescence microscopy was performed using anti PDI ( green ) as an ER marker . Detection of Cx32 was performed using anti FLAG antibodies suitable labeled secondary antibodies ( red ) . Nuclei were stained with DAPI ( blue ) . three channels are overlayed . are representative of from at least three different biological replicates . GJ denotes gap junctions observed at - junctions of transfected with WT Cx32 .

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F Immunofluorescence images of Cx32 and its mutants. COS-7 cells were transiently transfected with the indicated FLAG-tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). All three channels are overlayed. Images are representative of cells from at least three different biological replicates. GJ denotes gap junctions observed at cell-cell junctions of cells transfected with WT Cx32.


B Co - immunoprecipitations of endogenous CNX with different CL - TMD constructs ( NVT glycosylation site mutated to QVT ) including statistical . One representative immunoblot is shown . asterisk ( * ) denotes a fraction of species as observed previously for similar constructs ( Behnke et al . , 2016 ) . samples were normalized to construct containing

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B Co-immunoprecipitations of endogenous CNX with the different CL-TMD constructs (NVT glycosylation site mutated to QVT) including quantification and statistical analysis. One representative immunoblot is shown. The asterisk (*) denotes a fraction of cleaved species as observed previously for similar constructs (Behnke et al., 2016). All samples were normalized to the construct containing TMD1


C Interaction of Calnexin with first transmembrane domain of Cx32 . Intracellular BMH crosslinking of transiently transfected V5 - tagged CNX with transiently transfected R26C mutant of Cx32 - TMD1 . In AA mutant of CNX , two cysteines within CNX TMD were replaced by alanines . species at approx . 250 kDa are possibly CNX dimers .

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C Interaction analysis of Calnexin with the first transmembrane domain of Cx32. Intracellular BMH crosslinking of transiently transfected V5-tagged CNX with the transiently transfected R26C mutant of Cx32-TMD1. In the AA mutant of CNX, the two cysteines within the CNX TMD were replaced by alanines. The species at approx. 250 kDa are possibly CNX dimers.


E COS - 7 were transiently transfected with indicated constructs immunofluorescence microscopy was performed using anti PDI ( green ) as an ER marker . Detection of minCNXTMD both control constructs was carried out by mScarlet - I ( red ) . Nuclei were stained with DAPI ( blue ) . are representative of from at least three different biological replicates .

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E COS-7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of minCNXTMD and both control constructs was carried out by mScarlet-I fluorescence (red). Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.


F Interaction of Cx32 - TMD1 with minCNX system . MinCNXTMD constructs controls ( see ( D ) ) were co - transfected with Cx32 - TMD1 into HEK293T . Interaction between was analyzed by co - immunoprecipitation experiments followed by immunoblotting .

F Interaction of Cx32-TMD1 with the minCNX system. MinCNXTMD constructs and controls (see (D)) were co-transfected with Cx32-TMD1 into HEK293T cells. Interaction between the proteins was analyzed by co-immunoprecipitation experiments followed by immunoblotting.


B HEK293T were transiently transfected with plasmids expressing ConMem or indicated variants , where either first ( N1 ) or second ( N2 ) NVT glycosylation motif was ablated ( N to Q mutation ) . Lysates were treated with or without EndoH or PNGaseF as indicated , analyzed by immunoblotting . N - glycosylation occurs in ER , glycosylation in Golgi .

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B HEK293T cells were transiently transfected with plasmids expressing ConMem or the indicated variants, where either the first (N1) or the second (N2) NVT glycosylation motif was ablated (N to Q mutation). Lysates were treated with or without EndoH or PNGaseF as indicated, and analyzed by immunoblotting. N-glycosylation occurs in the ER, complex glycosylation in the Golgi.


C COS - 7 were transfected with indicated constructs microscopy was performed using Sec61 mCherry ( red ) as an ER marker . ConMem , detected by sfGFP ( green ) , localizes to plasma . Nuclei were stained with DAPI ( blue ) . are representative of from at least three different biological replicates .

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C COS-7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry (red) as an ER marker. ConMem, detected by sfGFP fluorescence (green), localizes to the plasma membrane. Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.


B Representative blots from co - immunoprecipitation experiments from HEK293T transfected with indicated ConMem constructs with or without ER - lumenal glycosylation site ( N1 ) ( mean ± , N = 3 , * P value < 0 . 05 , two - Student ' s t ) . values were normalized to ConMem N1Q ( Val ) values that were set to 1 .

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B Representative blots from co-immunoprecipitation experiments from HEK293T cells transfected with the indicated ConMem constructs with or without ER-lumenal glycosylation site (N1) (mean ± SEM, N = 3, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.


C Interaction of ConMem N1Q variants as described in ( A ) with endogenous CNX ( mean ± , N ≥ 5 , * P value < 0 . 05 , two - Student ' s t ) . values were normalized to ConMem N1Q ( Val ) values that were set to 1 . D Same as in ( C ) only that selected ( proline arginine ) where shifted through ConMem N1Q TMD segment to five different positions as shown in illustration , revealing positional binding dependencies ( mean ± , N ≥ 4 , * P value < 0 . 05 , two - Student ' s t ) . values were normalized to ConMem N1Q ( Val ) values that were set to 1 .

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C Interaction of ConMem N1Q variants as described in (A) with endogenous CNX (mean ± SEM, N ≥ 5, *P value < 0.05, two-tailed Student's t tests ). Individual values were normalized to ConMem N1Q (Val) values that were set to 1. D Same as in (C) only that selected amino acids (proline and arginine) where shifted through the ConMem N1Q TMD segment to five different positions as shown in the illustration, revealing positional binding dependencies (mean ± SEM, N ≥ 4, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.


D Interaction of non - glycosylated ConMem N1Q constructs with V5 - tagged CNX . In CNX variants , important for interaction as revealed in ( A ) were replaced by valine residues . HEK293T were transiently transfected with indicated constructs lysates HA - immunoprecipitates were analyzed for HA - tagged ConMem N1Q co - immunoprecipitating CNX mutants . E same as in ( D ) for Cx32 - TMD1 F of mutations in CNX TMD region their effect on binding glycosylated ConMem . G same as in ( D ) for Cx32 full -

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D Interaction of non-glycosylated ConMem N1Q constructs with V5-tagged CNX. In CNX variants, amino acids important for interaction as revealed in (A) were replaced by valine residues. HEK293T cells were transiently transfected with the indicated constructs and cell lysates and HA-immunoprecipitates were analyzed for HA-tagged ConMem N1Q and co-immunoprecipitating CNX mutants. E The same as in (D) for Cx32-TMD1 F Analysis of mutations in the CNX TMD region and their effect on binding glycosylated ConMem. G The same as in (D) for Cx32 full-length


H Interaction of Cx32 - TMD1 with CNX mutants , where not predicted to be important for interaction as shown in ( A ) were exchanged by valine residues . HEK293T were transiently transfected with indicated constructs .

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H Interaction of Cx32-TMD1 with CNX mutants, where amino acids not predicted to be important for interaction as shown in (A) were exchanged by valine residues. HEK293T cells were transiently transfected with the indicated constructs.


I Shifting of residues Y , T L , most important vor CNX substrate interaction , replacing these against valine residues , impacts interactions with Cx32 - TMD1 . HEK293T were transiently transfected with indicated constructs .

I Shifting of the residues Y, T and L, most important vor CNX substrate interaction, and replacing these against valine residues, impacts interactions with Cx32-TMD1. HEK293T cells were transiently transfected with the indicated constructs.


A of interaction of binding deficient CNX YTL TMD variant with Rhodopsin . CNX knockout HEK293T were transiently transfected with either N - terminal ALFA - tagged Rhodopsin alone or in combination with indicated V5 - tagged CNX constructs . In absence of any CNX , Rho is hardly detectable ( long exposure shown ) . lysates ALFA - immunoprecipitates were analyzed for Rhodopsin co - immunoprecipitating CNX .

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A Analysis of the interaction of the binding deficient CNX YTL TMD variant with Rhodopsin. CNX knockout HEK293T cells were transiently transfected with either N-terminal ALFA-tagged Rhodopsin alone or in combination with the indicated V5-tagged CNX constructs. In the absence of any CNX, Rho is hardly detectable (long exposure shown). Cell lysates and ALFA-immunoprecipitates were analyzed for Rhodopsin and co-immunoprecipitating CNX.


B of Rhodopsin degradation its dependency on CNX . CNX knockout HEK293T that were transfected with indicated constructs were incubated with cycloheximide lysates were collected at different timepoints . CNX WT CNX YTL immunoblots are from same blot ( dashed ) , showing similar expression . Hsc70 was used as a loading control ( also from same blot , dashed ) . Quantifications are shown below representative

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

B Analysis of Rhodopsin degradation and its dependency on CNX. CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints. CNX WT and CNX YTL immunoblots are from the same blot (dashed line), showing similar expression levels. Hsc70 was used as a loading control (also from the same blot, dashed line). Quantifications are shown below representative immunoblots


( C , D ) of SCOT BDH1 expression relative to VCP in various murine organs . Data are presented as mean ± SD . n = 3 ; * , p < 0 . 05 ; * * * , p < 0 . 001 unpaired Student ' s t - .

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(C, D) Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs. Data are presented as mean ± SD. n=3; *, p<0.05; ***, p<0.001 unpaired Student's t-test.


( G ) Immunoblot of SCOT , BDH1 , CD31 VCP in cardiac endothelial ( EC ) non - endothelial fraction ( non - EC ) isolated from adult C57Bl / 6J mice .

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(G) Immunoblot of SCOT, BDH1, CD31 and VCP in cardiac endothelial cells (EC) and non-endothelial cell fraction (non-EC) isolated from adult C57Bl/6J mice.


( A , B ) Murine cardiac endothelial ( MCEC ) were treated with 2 mM D - β - hydroxybutyrate ( β - OHB ) or 2 mM acetoacetate ( AcAc ) for 24 hours . of concentrations of TCA cycle intermediates compared to control treatment . Data are presented as mean ± SD . n ≥ 4 ; ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - .

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

(A, B) Murine cardiac endothelial cells (MCEC) were treated with 2 mM D-β-hydroxybutyrate (β-OHB) or 2 mM acetoacetate (AcAc) for 24 hours. Quantification of concentrations of TCA cycle intermediates compared to control treatment. Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( D ) Quantifications of [ 13C ] - labeled fractions of ( asparagine , L - aspartate , L - glutamate , L - proline ) , putative lipid species ( nervonic acid , 1 - palmitoylglycerol , ceramide ( d18 : 1 / 16 : 0 ) ) uridine diphosphate ( UDP ) species ( uridine monophosphate , uridine 5 ' - diphosphate , UDP - N - acetylglucosamine ) in MCEC treated with 1 mM 13C4 - β - hydroxybutyrate or 1 mM 13C4 - acetoacetate for 24 hours or same of unlabeled ketone bodies ( [ 12C ] isotope ) . Data are presented as mean ± SD . n ≥ 4 ; ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - .

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

(D) Quantifications of [13C]-labeled fractions of amino acids (asparagine, L-aspartate, L-glutamate, L-proline), putative lipid species (nervonic acid, 1-palmitoylglycerol, ceramide (d18:1/16:0)) and uridine diphosphate (UDP) species (uridine monophosphate, uridine 5'-diphosphate, UDP-N-acetylglucosamine) in MCEC treated with 1 mM 13C4-β-hydroxybutyrate or 1 mM 13C4-acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( A , B ) Murine cardiac endothelial ( MCEC ) were treated with different concentrations of D - β - hydroxybutyrate ( βOHB ) or acetoacetate ( AcAc ) for 24 hours . Relative BrdU absorbance was quantified compared to control treatment ( H2O for βOHB EtOH for AcAc ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - .

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

(A, B) Murine cardiac endothelial cells (MCEC) were treated with different concentrations of D-β-hydroxybutyrate (βOHB) or acetoacetate (AcAc) for 24 hours. Relative BrdU absorbance was quantified compared to control treatment (H2O for βOHB and EtOH for AcAc). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( I - M ) Angiogenic capacity of MCEC in response to ketone treatment was analyzed using a spheroid - sprouting . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) , 10 mM acetate , 1 mM octanoate , 1 mM butyrate , 30 mM L - β - hydroxybutyrate ( L - βOHB ) , 1 mM niacin or respective controls ( final of reagents is diluted to approximately 10 % ) analyzed after 48 hours . ( I , L ) average number of sprouts per spheroid , ( J , M ) accumulated total sprout ( K ) average sprout were quantified . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - .

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

(I-M) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc), 10 mM acetate, 1 mM octanoate, 1 mM butyrate, 30 mM L-β-hydroxybutyrate (L- βOHB), 1 mM niacin or the respective controls (final concentration of reagents is diluted to approximately 10 %) and analyzed after 48 hours. The (I, L) average number of sprouts per spheroid, the (J, M) accumulated total sprout length and (K) average sprout length were quantified. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( B , Angiogenic capacity of Oxct1 knockout in response to ketone treatment was analyzed using a spheroid - sprouting . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) or respective controls ( final of reagents is diluted to approximately 10 % ) for 48 hours . average number of sprouts per spheroid was quantified . Data are presented as mean ± SD . n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; unpaired Student ' s t - .

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(B, Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of sprouts per spheroid was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.


( C , D ) Heatmaps showing expression of genes involved in KEGG pathways ( C ) fatty acid degradation ( D ) ketone metabolism in cardiac endothelial isolated from C57Bl / 6J mice that were fed a ketogenic diet or control diet for two .

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(C, D) Heatmaps showing expression levels of genes involved in KEGG pathways (C) fatty acid degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice that were fed a ketogenic diet or control diet for two weeks.


( A , B ) Representative of heart sections of kept on a ketogenic diet or control diet for two stained against ERG Ki67 or EdU , double positive are indicated by arrowheads , of double positive in heart sections per high power field ( HPF ) . Scale bar : 50 μm . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - .

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(A, B) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU, double positive cells are indicated by arrowheads, and quantification of double positive cells in heart sections per high power field (HPF). Scale bar: 50 μm. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( F ) of Ki67 + endothelial in several organs of mice kept on a ketogenic diet or control diet for four ( gastrocnemius soleus muscle ) or two ( brain , lungs , subcutaneous adipose tissue , liver ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - .

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(F) Quantification of Ki67+ endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks (gastrocnemius and soleus muscle) or two weeks (brain, lungs, subcutaneous adipose tissue, liver). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


( B ) Representative of heart sections of mice kept on a control or ketogenic diet eight after TAC surgery stained against Ki67 ERG . Scale bar : 50 µm .

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(B) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG. Scale bar: 50 µm.


( A - C ) Tissue lysates from ( A ) spleen , ( B ) liver ( C ) kidney from 6 - to 9 - day old indicated mice were immunoblotted to determine cleavage of caspase - 1 . Each lane represents a lysate from a different mouse . Positive control lanes ( + ) are BMDMs stimulated with LPS / ATP ; negative control lanes ( - ) are respective organ homogenates from caspase - 1 - / - mice .

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(A-C) Tissue lysates from (A) spleen, (B) liver and (C) kidney from 6- to 9-day old indicated mice were immunoblotted to determine cleavage of caspase-1. Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from caspase-1-/- mice.


( D - G ) Tissue lysates from ( D , F ) spleen ( E , G ) liver from 6 - to 9 - day old indicated mice were immunoblotted to determine pro - IL - 1β cleavage to IL - 1β ( D - E ) or GSDMD cleavage to its p30 p20 fragments ( F - G ) . Each lane represents a lysate from a different mouse . Positive control lanes ( + ) are BMDMs stimulated with LPS / ATP ; negative control lanes ( - ) are respective organ homogenates from ( D - E ) caspase - 1 - / - or ( F - G ) Gsdmd - / - mice .

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(D-G) Tissue lysates from (D, F) spleen and (E, G) liver from 6- to 9-day old indicated mice were immunoblotted to determine pro-IL-1β cleavage to IL-1β (D-E) or GSDMD cleavage to its p30 and p20 fragments (F-G). Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from (D-E) caspase-1-/- or (F-G) Gsdmd-/- mice.


Coomassie - stained from bovine retina that were co - immunoprecipitated by anti - RIBEYE antibody 2D9 ( lane 3 ) . Lane 2 , control precipitation by irrelevant mouse IgGs ; lane 1 , input ( 10 % of total loaded in each lane ) Western blot of immunoprecipitated ( as in A , 1 % of total was loaded per lane ) probed with anti - RIBEYE ( Ba ) , anti - CASPR1 ( Bb ) anti - CNTN1 ( Bc ) . 2D9 antibody immunoprecipitated RIBEYE ( Ba , lane 3 ) , CASPR1 ( Bb , lane 3 ) CNTN1 ( Bc , lane 3 ) ; these were not detectable in control immunoprecipitations . Bd ) As a loading control , heavy chains of precipitating antibody ( IgG hc ) were visualized by goat anti - mouse IgG antibody conjugated to

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Coomassie- stained proteins from bovine retina that were co-immunoprecipitated by the anti-RIBEYE antibody 2D9 (lane 3). Lane 2, control precipitation by irrelevant mouse IgGs; lane 1, input (10% of the total loaded in each lane) Western blot of the immunoprecipitated proteins (as in A, 1% of the total protein was loaded per lane) probed with anti-RIBEYE (Ba), anti-CASPR1 (Bb) and anti-CNTN1 (Bc). The 2D9 antibody immunoprecipitated RIBEYE (Ba, lane 3), CASPR1 (Bb, lane 3) and CNTN1 (Bc, lane 3); these proteins were not detectable in the control immunoprecipitations. Bd) As a loading control, heavy chains of the precipitating antibody (IgG hc) were visualized by the goat anti-mouse IgG antibody conjugated to peroxidase


Localization of CASPR in semi - thin sections of retinas from either MOG / CFA - injected mice ( EAE mice ) or CFA - injected control mice ( 9 days after injection ) . Semi - thin sections were double - immunolabelled with antibodies against CASPR1 RIBEYE ( A - B OPL , outer plexiform layer ; INL , layer ; IPL , inner plexiform layer . Scale bars :

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Localization of CASPR in semi-thin sections of retinas from either MOG/CFA-injected mice (EAE mice) or CFA-injected control mice (9 days after injection). Semi-thin sections were double-immunolabelled with antibodies against CASPR1 and RIBEYE (A-B OPL, outer plexiform layer; INL, nuclear layer; IPL, inner plexiform layer. Scale bars: 20μm