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 .

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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 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 .

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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 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 .

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

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 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 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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 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 ) .

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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 . 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 .

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

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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 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

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

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

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 . 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 .

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

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 . 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 .

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

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 . 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 .

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

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 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 ) .

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

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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 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 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR 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 ) 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

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 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 .

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

(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 ) 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 ) .

O O O O O O O O O B-MEASURED_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 B-MEASURED_VAR 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 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 ) .

(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 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 .

O O O O O O O O O O O O O O O O O O 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 + / + 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 .

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

(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 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 .

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(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 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 .

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

(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 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 .

O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O 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 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 .

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

(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 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 .

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

(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 + / + 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 .

O O O O O B-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 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 .

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(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 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 .

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(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 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 .

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

(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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 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 .

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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 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 .

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(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 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 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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 , 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 .

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(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 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

O O O O O O O O O O O O O O O O O O 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) 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 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 .

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(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 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 .

(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 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 .

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(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 ) 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 .

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(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 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 .

(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 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 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 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 .

(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 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 .

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

(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 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 .

(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 - / - 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 .

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(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 ) , 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 .

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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 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 ) .

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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 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 ) .

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

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(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 ) 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

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(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 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 .

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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 ± 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 .

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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 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 ) .

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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 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 .

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(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 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 ) .

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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 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 .

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(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 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 ) .

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(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 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 .

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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 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

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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 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 .

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(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 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 ) .

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(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 ) 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 ) .

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(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 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 ) .

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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 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 .

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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 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 .

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(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 and IFNλ3 ) pro - inflammatory mediators ( IL - 6 and 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 and 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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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(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 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 .

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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 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 .

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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 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 .

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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 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

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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 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 .

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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 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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ± 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 .

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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 , 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 -

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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 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 .

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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 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 .

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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 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 .

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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 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

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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 ) 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 .

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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 and VCP in cardiac endothelial cells ( EC ) and non - endothelial cell 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 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 .

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(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 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 .

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(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 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 .

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(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.