E . ITC assays of HY5 UVR8 VP - peptides versus different COP1 WD40 domain mutants following concentrations were typically used ( titrant into ) : UVR8 - COP1Lys422Ala ( 1000 μM in 100 μM ) ; UVR8 - COP1Tyr441Ala ( 2500 μM in 90 μM ) ; UVR8 - COP1Trp467Ala ( 2500 μM in 90 μM ) ; HY5 - COP1Lys422Ala ( 1500 μM in 175 μM ) ; HY5 - COP1Tyr441Ala ( 1600 μM in 138 μM ) ; HY5 - COP1Trp467Ala ( 1600 μM in 112 μM ) . insets show dissociation constants ( Kd ) . stoichiometries of binding ( N ) for UVR8 - COP1Lys422Ala = 0 . 87 ± 0 . 2 ; HY5 - COP1Lys422Ala = 0 . 83 ± 0 . 2 ; HY5 - COP1Tyr441Ala = 0 . 92 ± 0 . 3 ( measurements ± standard deviation ; n . d . : no detectable binding ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

E. ITC assays of the HY5 and UVR8 VP-peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used (titrant into cell): UVR8 - COP1Lys422Ala (1000 μM in 100 μM); UVR8 - COP1Tyr441Ala (2500 μM in 90 μM); UVR8 - COP1Trp467Ala (2500 μM in 90 μM); HY5 - COP1Lys422Ala (1500 μM in 175 μM); HY5 - COP1Tyr441Ala (1600 μM in 138 μM); HY5 - COP1Trp467Ala (1600 μM in 112 μM). The insets show the dissociation constants (Kd). The stoichiometries of binding (N) for UVR8 - COP1Lys422Ala = 0.87 ± 0.2; HY5 - COP1Lys422Ala = 0.83 ± 0.2; HY5 - COP1Tyr441Ala = 0.92 ± 0.3 (All measurements ± standard deviation; n.d.: no detectable binding).


of representative individuals ( F ) of 4 - day - old seedlings grown with or without supplemental UV - B . scale bar for lines represent 5 mm except for cop1 - 5 where scale bars represent 1 mm . Data Information , lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala cop1 - 5 . # 1 # 2 : independent transgenic lines .

O O O O O O O O O O O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

Images of representative individuals (F) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Data Information , lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


of hypocotyl lengths ( G ) of 4 - day - old seedlings grown with or without supplemental UV - B . scale bar for lines represent 5 mm except for cop1 - 5 where scale bars represent 1 mm . Violin box plots are shown for n > 60 seedlings ; upper lower hinges correspond to first third quartiles , horizontal in interior of box indicates median . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala cop1 - 5 . # 1 # 2 : independent transgenic lines .

O B-TISSUE B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O 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-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

quantification of hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


Quantitative real - time PCR of ( H ) HY5 expression . Four - day - old seedlings grown in were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent of 3 biological replicates . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala cop1 - 5 . # 1 # 2 : independent transgenic lines .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

Quantitative real-time PCR analysis of (H) HY5 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


Quantitative real - time PCR of ( I ) RUP2 expression . Four - day - old seedlings grown in were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent of 3 biological replicates . Data Information lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala cop1 - 5 . # 1 # 2 : independent transgenic lines .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

Quantitative real-time PCR analysis of (I) RUP2 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


A . ITC between COP1 WD40 domain full - UVR8 pre - monomerized by UV - B . Integrated heats are shown in solid , purple squares . For comparison , an ITC experiment between UVR8 VP peptide COP1 WD40 domain ( from Fig 1B ) is also shown in open , purple squares . following concentrations were typically used ( titrant into ) : COP1 - UVR8 + UV - B ( 130 μM in 20 μM ) . inset shows dissociation constant ( Kd ) , stoichiometry of binding ( N ) ( ± standard deviation ) .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

A. ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1 - UVR8 +UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation).


B . Binding kinetics of UVR8 pre - monomerized by UV - B versus COP1 WD40 domain obtained by grating - coupled interferometry ( GCI ) . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8 + UV - B ( highest 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O 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. Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


D . Binding kinetics of UVR8ValPro / AlaAla pre - monomerized by UV - B versus COP1 WD40 domain obtained by GCI experiments . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8ValPro / AlaAla + UV - B ( highest 2 μM ) . ka = association rate constant , kd = disassociation rate constant , Kd = dissociation constant .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O 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. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.


E . Binding kinetics of HY5 versus COP1 WD40 domain obtained by GCI experiments . Sensorgrams of HY5 injected are shown in red , with their respective 1 : 1 binding model fits in black . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - HY5 ( highest 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

E. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


F . Yeast 3 - hybrid of COP1 - HY5 interaction in presence of UVR8 . ( Top ) Normalized Miller Units were calculated as a of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means for 3 biological repetitions are shown . ( Bottom ) representative filter lift assays . AD , activation domain ; BD , binding domain ; Met , methionine .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.


B . Representative image showing phenotype of wild - type ( Ws ) , uvr8 - 7 uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP uvr8 - 7 / Pro35S : UVR8TRIB1 seedlings grown for 4 days under ( - UV - B ) or supplemented with UV - B ( + UV - B ) . scale bar represents 5 mm .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD B-ORGANISM O O O O O O 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. Representative image showing the phenotype of wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 seedlings grown for 4 days under white light (- UV-B) or white light supplemented with UV-B (+ UV-B). The scale bar represents 5 mm.


C . of hypocotyl data Violin box plots are shown for n > 60 seedlings ; upper lower hinges correspond to first third quartiles , horizontal in interior of box indicates median .

O O O B-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

C. Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median.


D . Anthocyanin accumulation in seedlings Average are shown ( n = 3 ) .

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D. Anthocyanin accumulation in seedlings Average and SEM are shown (n = 3).


E . Quantitative real - time PCR of RUP2 , CHS , HY5 ELIP2 expression in wild - type ( Ws ) , uvr8 - 7 uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP uvr8 - 7 / Pro35S : UVR8TRIB1 4 - day old seedlings grown under in response to 2h of UV - B ( + UV - B vs . - UV - B ) . Error bars represent of 3 biological replicates . Note : primers used to detect HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O 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-GENEPROD O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O

E. Quantitative real-time PCR analysis of RUP2, CHS, HY5 and ELIP2 expression in wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 4-day old seedlings grown under white light in response to 2h of UV-B (+UV-B vs. -UV-B). Error bars represent SEM of 3 biological replicates. Note: the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera.


F . Yeast 3 - hybrid of COP1 - HY5 interaction in presence of UVR8HY5C44 chimera . ( Left pane ) Normalized Miller Units were calculated as a of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means for 3 biological repetitions are shown . ( Right panel ) Representative filter lift assays of yeast analyzed in left panel . AD , activation domain ; BD , binding domain ; Met , methionine .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

F. Yeast 3-hybrid analysis of the COP1-HY5 interaction in the presence of the UVR8HY5C44 chimera. (Left pane) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Right panel) Representative filter lift assays of the yeast analyzed in left panel. AD, activation domain; BD, DNA binding domain; Met, methionine.


G . Binding kinetics of UVR8HY5C44 chimera pre - monomerized by UV - B versus COP1 WD40 domain obtained by GCI experiments . Sensorgrams of UVR8HY5C44 injected are shown in red , with their respective 1 : 1 binding model fits in black . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8HY5C44 + UV - B ( highest 2 μM ) . ka = association rate constant , kd = disassociation rate constant , Kd = dissociation constant .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O 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. Binding kinetics of the UVR8HY5C44 chimera pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8HY5C44 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8HY5C44 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.


H . - exclusion chromatography of purified , recombinant chimeric expressed in Sf9 insect in presence absence of UV - B .

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H. Size-exclusion chromatography assay of purified, recombinant chimeric proteins expressed in Sf9 insect cells in the presence and absence of UV-B.


A . Table summarizing affinities ( Kd , dissociation constant ) of VP - containing peptides versus COP1 WD40 domain ( WT ) COP1Lys422Ala as determined by ITC . values are μM ( ± standard deviation ) .

O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O

A. Table summarizing affinities (Kd, dissociation constant) of VP-containing peptides versus the COP1 WD40 domain (WT) and COP1Lys422Ala as determined by ITC. All values are μM (± standard deviation).


D . ITC assays between COL3 VP - peptide ( left ) CRY1 VP - peptide ( right ) versus COP1 WD40 COP1Lys422Ala , with a table summarizing their corresponding affinities . following concentrations were typically used ( titrant into ) : COL3 - COP1 ( 2240 μM in 195 μM ) ; COL3 - COP1Lys422Ala ( 1500 μM in 175 μM ) ; CRY1 - COP1 ( 750 μM in 75 μM ) ; CRY1 - COP1Lys422Ala ( 1500 μM in 175 μM ) . inset shows dissociation constant ( Kd ) . stoichiometries of binding ( N ) for COL3 - COP1 = 0 . 75 ± 0 . 2 ; COL3 - COP1Lys422Ala = 0 . 90 ± 0 . 2 ; CRY1 - COP1 = 0 . 99 ± 0 . 3 ; CRY1 - COP1Lys422Ala = 0 . 98 ± 0 . 1 ( measurements ± standard deviation ) .

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D. ITC assays between the COL3 VP-peptide (left) and CRY1 VP-peptide (right) versus the COP1 WD40 and COP1Lys422Ala, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): COL3 - COP1 (2240 μM in 195 μM); COL3 - COP1Lys422Ala (1500 μM in 175 μM); CRY1 - COP1 (750 μM in 75 μM); CRY1 - COP1Lys422Ala (1500 μM in 175 μM). The inset shows the dissociation constant (Kd). The stoichiometries of binding (N) for COL3 - COP1 = 0.75 ± 0.2; COL3 - COP1Lys422Ala = 0.90 ± 0.2; CRY1 - COP1 = 0.99 ± 0.3; CRY1 - COP1Lys422Ala = 0.98 ± 0.1 (All measurements ± standard deviation).


A . Representative image of wild - type ( Ws ) , cop1 - 5 / Pro35S : YFP - COP1 , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala cop1 - 5 / Pro35S : YFP - COP1Trp467Ala transgenic lines grown for 39 days in long day conditions .

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A. Representative image of wild-type (Ws), cop1-5/Pro35S:YFP-COP1, cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala and cop1-5/Pro35S:YFP-COP1Trp467Ala transgenic lines grown for 39 days in long day conditions.


B . Representative image of wild - type ( Ws ) , co - 11 , cop1 - 5 / Pro35S : YFP - COP1 cop1 - 5 / Pro35S : YFP - COP1Lys422Ala grown for 39 days in long days conditions .

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B. Representative image of individual wild-type (Ws), co-11, cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala plants grown for 39 days in long days conditions.


C . of flowering time . Means SD are shown ( n = 14 ) . D . Number of rosette cauline leaves at flowering . Means SD are shown ( n = 14 ) .

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C. Quantification of flowering time. Means and SD are shown (n = 14). D. Number of rosette and cauline leaves at flowering. Means and SD are shown (n = 14).


E . Immunoblot of CRY2 actin ( loading control ) in wild - type ( Col ) , cry2 - 1 , wild - type ( Ws ) , cop1 - 5 / Pro35S : YFP - COP1 cop1 - 5 / Pro35S : YFP - COP1Lys422Ala seedlings grown for 4 days in darkness .

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E. Immunoblot analysis of CRY2 and actin (loading control) protein levels in wild-type (Col), cry2-1, wild-type (Ws), cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala seedlings grown for 4 days in darkness.


F . ITC assays between CO VP - peptide ( left ) CRY2 VP - peptide ( right ) versus COP1 WD40 COP1Lys422Ala WD40 domains , with a table summarizing their corresponding affinities . following concentrations were typically used ( titrant into ) : CO - COP1 ( 2240 μM in 195 μM ) ; CO - COP1Lys422Ala ( 1200 μM in 120 μM ) ; CRY2 - COP1 ( 750 μM in 70 μM ) ; CRY2 - COP1Lys422Ala ( 3000 μM in 175 μM ) . stoichiometries of binding ( N ) for CO - COP1 = 1 . 03 ± 0 . 00 ; CO - COP1Lys422Ala = 0 . 89 ± 0 . 03 ; CRY2 - COP1 = 1 . 04 ± 0 . 20 ; CRY2 - COP1Lys422Ala = 1 ( fixed ) ( measurements ± standard deviation ) .

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F. ITC assays between the CO VP-peptide (left) and CRY2 VP-peptide (right) versus the COP1 WD40 and the COP1Lys422Ala WD40 domains, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): CO - COP1 (2240 μM in 195 μM); CO - COP1Lys422Ala (1200 μM in 120 μM); CRY2 - COP1 (750 μM in 70 μM); CRY2 - COP1Lys422Ala (3000 μM in 175 μM). The stoichiometries of binding (N) for CO - COP1 = 1.03 ± 0.00; CO - COP1Lys422Ala = 0.89 ± 0.03; CRY2 - COP1 = 1.04 ± 0.20; CRY2 - COP1Lys422Ala = 1 (fixed) (All measurements ± standard deviation).


G , H . Binding kinetics of ( G ) full - activated CRY2 ( H ) CRY2NC80 versus COP1 WD40 domain obtained by GCI experiments . Sensorgrams of injected are shown in red , with their respective heterogenous ligand binding model fits in black . following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - CRY2 ( highest 14 μM ) , CRY2NC80 ( highest 450 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

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G, H. Binding kinetics of the (G) full-length activated CRY2 and (H) CRY2NC80 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of protein injected are shown in red, with their respective heterogenous ligand binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - CRY2 (highest concentration 14 μM), CRY2NC80 (highest concentration 450 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


A ) Western blot of Mpl in healthy donors ' ( HD ) platelets shows two distinct bands representing mature glycoprotein ( top band ) ER ‐ glycosylated form ( bottom band ) . is ~ 1 .

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A) Western blot analysis of Mpl in healthy donors' (HD) platelets shows two distinct bands representing the mature glycoprotein (top band) and the ER‐core glycosylated form (bottom band). The ratio is ~ 1.


B ) Western blot of HEL whole lysate after transfection with cDNA coding for JAK2WT or JAK2V617F , alone or cotransfected with cDNA coding for Mpl .

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B) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F, alone or cotransfected with cDNA coding for Mpl.


C ) Summary of ratios of mature : immature Mpl for at least three independent ( 2 for JAK2V617F transfection ) blotting experiments under protocol shown in ( B ) . on two − Student t − statistical , there is a significant difference ( p = 0 . 0001 ) in of Mpl isoforms in transfected with JAK2WT by comparison with control . Differences were also statistically significant in HEL transfected with either JAK2V617F or JAK2L611V / V617F by comparison to controls ( p = 0 . 0159 p = 0 . 0009 , respectively ) . mature : immature is also significantly higher in transfected with JAK2WT than in transfected with JAK2V617F or JAK2L611V / V617F ( p < 0 . 01 ) .

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C) Summary of the ratios of mature:immature Mpl for at least three independent (2 for the JAK2V617F transfection) blotting experiments under the protocol shown in (B). Based on two−tailed Student t−test statistical analysis, there is a significant difference (p = 0.0001) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells. Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V/V617F by comparison to controls (p = 0.0159 and p = 0.0009, respectively). The mature:immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V/V617F (p < 0.01).


D ) Western blot of K562 lysates after transfection with cDNA coding for Mpl , alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F .

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D) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl, alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F.


E ) Summary of data in ( A ) , ( B ) ( D ) , showing relative band of mature immature Mpl normalized for of β ‐ actin in each lane .

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E) Summary of data in (A), (B) and (D), showing the relative band intensity of mature and immature Mpl and normalized for levels of β‐actin in each lane.


F ) Immunoprecipitation of Mpl ‐ Jak2 in different lines analyzed by immunoblotting with anti ‐ Mpl anti ‐ JAK2 antibodies .

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F) Immunoprecipitation of the Mpl‐Jak2 complex in different cell lines analyzed by immunoblotting with anti‐Mpl and anti‐JAK2 antibodies.


Confocal of HEL ( JAK2V617F + / + ) after immuno ‐ labeling of endogenous Mpl ( A ) , or after transient transfection with cDNA coding for a chimeric Mpl fused to mOrange2 ( B ) .

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Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno‐fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B).


C ) Confocal image of K562 ( JAK2WT + / + ) transiently transfected with cDNA coding for MplmOrange2 . Red arrow in this image points to plasma localization .

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C) Confocal fluorescence image of K562 cells (JAK2WT+/+) transiently transfected with cDNA coding for MplmOrange2. Red arrow in this image points to plasma membrane localization.


D E ) Confocal obtained after transient expression of MplmOrange2 immuno ‐ labeling of endogenous JAK2 in K562 ( D ) or HEL ( E ) . Profiles of ( across indicated by a arrow ) are showing a good colocalization of Mpl JAK2WT in K562 at plasma ( black arrow heads ) but no colocalization have been observed between MplmOrange2 JAK2V617F in HEL .

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D and E) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno‐fluorescent labeling of endogenous JAK2 in K562 (D) or HEL (E) cells. Profiles of fluorescence (across individual cells and indicated by a white arrow) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane (black arrow heads) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL.


F G ) Confocal obtained after transient coexpression of MplmOrange2 either JAK2WTmCitrine ( F ) or JAK2V617FmCitrine ( G ) in K562 . Profiles of ( arrows ) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine , especially at plasma ( black arrow heads ) , but a very poor colocalization with JAK2V617FmCitrine ( grey arrow heads ) . Nuclei are labeled with Hoechst scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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F and G) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine (F) or JAK2V617FmCitrine (G) in K562 cells. Profiles of fluorescence (white arrows) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine, especially at the plasma membrane (black arrow heads), but a very poor colocalization with JAK2V617FmCitrine (grey arrow heads). Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


A ) K562 labeled live for 30 min with an anti ‐ CD235a ‐ Atto655 ( glycophorin A ) antibody before treatment with Duolink reagents .

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A) K562 cells labeled live for 30 min with an anti‐CD235a‐Atto655 (glycophorin A) antibody before treatment with the Duolink reagents.


B ) K562Mpl ‐ mKO2 non ‐ treated with Duolink reagents .

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B) K562Mpl‐mKO2 cells non‐treated with the Duolink reagents.


C D ) HEL primed live for 30 min with an anti ‐ CD235a ‐ Atto655 antibody before treatment with Duolink reagents .

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C and D) HEL cells primed live for 30 min with an anti‐CD235a‐Atto655 antibody before treatment with the Duolink reagents.


E F ) K562Mpl ‐ mKO2 submitted to Duolink reaction . arrow heads are pointing at example of Duolink positive reactions at plasma , while green arrow heads are pointing at positive Duolink reactions within cytoplasm . Nuclei are labeled with Hoechst scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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E and F) K562Mpl‐mKO2 cells submitted to the Duolink reaction. White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane, while green arrow heads are pointing at positive Duolink reactions within the cytoplasm. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


HEL ( A ) K562 ( B ) treated with non ‐ targeting ( NT ) or anti ‐ JAK2 siRNA analyzed by western blot .

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HEL (A) and K562 (B) cells treated with non‐targeting (NT) or anti‐JAK2 siRNA analyzed by western blot.


C ) Confocal live ( upper panel ) , or fixed ( lower left panel ) , imaging of K562 expressing MpleGFP or MplmKO2 treated with NT or anti ‐ JAK2 siRNA showing a difference of distribution of intracellular Mpl in siRNA treated compare to control . After treatment with anti ‐ JAK2 siRNA , Mpl strongly colocalizes with ER marker , calnexin . This colocalization in treated vs non ‐ treated is quantified shown in Figure C lower right panel . Nuclei are labeled with Hoechst scale bars = 5 µm .

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C) Confocal live cell (upper panel), or fixed cells (lower left panel), imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti‐JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells. After treatment with anti‐JAK2 siRNA, Mpl strongly colocalizes with the ER marker, calnexin. This colocalization in treated vs non‐treated cells is quantified and shown in Figure C lower right panel. Nuclei are labeled with Hoechst and scale bars = 5 µm.


Intracellular Mpl pools partially colocalize with ER markers colocalize with autophagic lysosomal markers in K562 HEL . A - M ) Confocal obtained after transient expression of MplmOrange2 or MplmKO2 indicated labeling in K562 ( A - F ) or HEL ( G - M ) . Plots at right of each image show intensities for each emission spectrum at points along drawn across each image . Black arrows in plots point to overlap between MplmKO2 either ER ‐ tracker ( C , I ) , or Sec31a ( B , J ) that marks ER exit sites . MplmOrange2 did not colocalize with markers for Golgi ( Golgin ‐ 97 ) or early endosomes ( EEA1 ) in either . However , MplmKO2 colocalized greatly with autophagic ( LC3 ) ( E , L ) lysosomal ( LAMP1 ) ( F , M ) markers . Nuclei are labeled with Hoechst scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


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Confocal obtained after transient expression of Mpl tagged with mKO2 miniSOG in K562 ( A ) HEL ( B ) . Electron of K562 ( C ) HEL ( D ) transiently expressing MplmKO2 ‐ miniSOG . precipitate indicates presence of miniSOG ‐ tagged Mpl after photo ‐ precipitation of DAB . boxes indicate original location of higher magnification with corresponding letters .

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Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 (A) and HEL (B) cells. Electron micrographs of K562 (C) and HEL (D) cells transiently expressing MplmKO2‐miniSOG. Dark precipitate indicates the presence of the miniSOG‐tagged Mpl protein after photo‐precipitation of DAB. White boxes indicate original location of higher magnification images with corresponding letters.


E - G ) High magnification electron micrograph of K562 treated in same conditions as Figure C , showing MplmKO2 ‐ miniSOG precipitate into autophagic carrier vesicles ( black arrows , Figure E ) . Osmiophilic reaction products also heavily stain mature AP in these preparations ( black arrows , Figure G ) .

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E-G) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C, showing MplmKO2‐miniSOG dark precipitate into autophagic carrier vesicles (black arrows, Figure E). Osmiophilic reaction products also heavily stain mature AP in these preparations (black arrows, Figure G).


H ) High magnification electron micrograph of HEL treated in same conditions as Figure D , transiently expressing MplmKO2 - miniSOG , showing different stage of formation of APs ( black arrows numbered from stages 1 - 4 ) . Scale bars = 5 µm ( A - D ) or 100 nm ( E - H ) .

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H) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D, cells transiently expressing MplmKO2-miniSOG , showing different stage of formation of APs (black arrows numbered from stages 1-4). Scale bars = 5 µm (A-D) or 100 nm (E-H).


A ) Western blot of streptavidin ‐ immunoprecipitated surface ‐ biotinylated , probed with an anti ‐ Mpl

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A) Western blot analysis and quantification of streptavidin‐immunoprecipitated surface‐biotinylated proteins, probed with an anti‐Mpl antibody


B C ) Confocal of K562 transiently expressing MplmOrange2 . in ( B ) are untreated . in ( C ) were stimulated 60 ' with Tpo , washed chased for 15 min before imaging .

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B and C) Confocal images of K562 cells transiently expressing MplmOrange2. Cells in (B) are untreated. Cells in (C) were stimulated 60' with Tpo, washed and chased for 15 min before imaging.


D ) of plasma in resting Tpo ‐ stimulated . Mean at was estimated by averaging total after values equal to or below background . A two ‐ Student t ‐ shows a significant difference in after stimulation with Tpo followed by Tpo removal . Scale bars = 5 µm .

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D) Quantification of plasma membrane fluorescence in resting and Tpo‐stimulated cells. Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background. A two‐tailed Student t‐test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal. Scale bars = 5 µm.


E ) Western blot of phosphorylation in HEL , K562Mpl ‐ mKO2 K562Mpl ‐ mOrange2 after Tpo stimulation ( 50 ng / mL Tpo , 10 min ) .

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E) Western blot analysis of protein phosphorylation in HEL, K562Mpl‐mKO2 and K562Mpl‐mOrange2 cells after Tpo stimulation (50 ng/mL Tpo, 10 min).


A B ) Surface biotinylation total lysates western blot of HEL or K562Mpl ‐ mKO2 after treatments for 60 min with Tpo , ionomycin ( I ) , PMA , Mon or A23187 .

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A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl‐mKO2 cells after treatments for 60 min with Tpo, ionomycin (I), PMA, Mon or A23187.


C ) Total lysate , surface biotinylation exosomal fraction western blot of HEL K562Mpl ‐ mKO2 treated with Mon or A23187 for 15 h .

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C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl‐mKO2 cells treated with Mon or A23187 for 15 h.


D ) Western blot of HEL treated , or not , with Bafilomycin A1 at 100 nm for 3 h then stimulated or not with Tpo at 25 ng / mL for 30 min . of blot is shown .

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D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng/mL for 30 min. The quantification of the blot is shown.


E ) Surface biotinylation total lysates western blot of HEL after treatments for 30 min with Tpo or for 3 h with 3 − MA ( 3 − Methyladenine ) or Rp .

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E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3−MA (3−Methyladenine) or Rp.


F ) Total lysate surface biotinylation western blot of HEL overexpressing ATG5 K130R ‐ V5 6 h post ‐ transfection .

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F) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R‐V5 6 h post‐transfection.


G ) Total lysate surface biotinylation western blot of HEL overexpressing either GRASP55 - V5 or HA - STX5 .

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G) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55-V5 or HA-STX5.


B , HeLa were treated with DMSO or 2µM Mito - C for 15 min immunostained with anti - TOM20 ( green ) antibody DAPI ; cropped areas show mitochondria changes .

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B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.


C , of mitochondria ( on TOM20 immunostaining as shown in B ) from HeLa treated with DMSO or 2µM Mito - C for 15 min or 2h , expressed as of fragmented mitochondria ( of total ) . Errors bars show standard error of mean ( ) ( n = 100 , technical replicates ) .

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C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).


E , Time lapse video - microscopy snapshot on HeLa transfected with NAF - 1 - mRFP ( red ) treated with fluoMito - C ( green ) . / graph illustrates codistribution of fluoMito - C NAF - 1 - mRFP .

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E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.


A , Western blot of MitoNEET expression in HeLa transfected with CISD1 siRNA ; Bar Chart ( right panel ) shows replicates of MitoNEET expression . Errors bars show standard deviation ( SD ) of 3 independent experiments .

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A, Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA; Bar Chart (right panel) shows replicates quantification of MitoNEET expression. Errors bars show the standard deviation (SD) of 3 independent experiments.


B , Western blot of NAF - 1 expression in HeLa transfected with CISD2 siRNA ; graph ( right panel ) shows replicates of NAF - 1 expression . Errors bars show standard deviation ( SD ) of 3 independent experiments .

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B, Western blot analysis of NAF-1 expression in HeLa cells transfected with CISD2 siRNA; graph (right panel) shows the replicates quantification of NAF-1 expression. Errors bars show the standard deviation (SD) of 3 independent experiments.


D , HeLa transfected with siRNA to reduce expression of MitoNEET ( CISD1 ) , NAF - 1 ( CISD2 ) or MiNT ( CISD3 ) immunostained with anti TOM20 antibody . Arrowheads indicate fragmented mitochondria .

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D, HeLa cells transfected with siRNA to reduce expression of MitoNEET (CISD1), NAF-1 (CISD2) or MiNT (CISD3) and immunostained with anti TOM20 antibody. Arrowheads indicate fragmented mitochondria.


E , of mitochondria ( on TOM20 immunostaining as shown in D ) from HeLa transfected with siRNA targeting mitoNEET ( siCISD1 ) , NAF1 ( siCISD2 ) or MiNT ( siCISD3 ) . is expressed as of tubular fragmented mitochondria . Errors bars show standard error of mean ( ) . ( n = 125 - 130 ) .

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E, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in D) from HeLa cells transfected with siRNA targeting mitoNEET (siCISD1), NAF1 (siCISD2) or MiNT (siCISD3). Quantification is expressed as ratio of tubular and fragmented mitochondria. Errors bars show the standard error of the mean (SEM). (n =125-130 cells).


F , Electron microscopy pictures from 150 INS - 1 transfected as described in G .

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F, Electron microscopy pictures from 150 INS-1 cells transfected as described in G.


G , Western blot of NAF - 1 expression in stably transfected with control shRNA , shRNA targeting reduction of NAF - 1 expression ( shCISD2 ) , or shRNA targeting reduction of NAF - 1 expression complemented with plasmid derived expression of NAF - 1 ( shCISD2 + CISD2 ) ; western blot of NAF - 1 is shown on accompanying bar chart . Errors bars show standard deviation ( SD ) of 3 independent experiments .

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G, Western blot analysis of NAF-1 expression in cells stably transfected with control shRNA, shRNA targeting reduction of NAF-1 protein expression (shCISD2), or shRNA targeting reduction of NAF-1 protein expression complemented with plasmid derived expression of NAF-1 (shCISD2+CISD2); western blot quantification of NAF-1 is shown on the accompanying bar chart. Errors bars show the standard deviation (SD) of 3 independent experiments.


H , of mitochondrial in EM from transfected as described in G . Errors bars show standard error of mean ( ) ( n = 260 - 270 , technical replicates ) .

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H, Quantification of mitochondrial length in EM images from cells transfected as described in G. Errors bars show the standard error of the mean (SEM) (n=260-270, technical replicates).


A , HeLa treated or not , with 2µM Mito - C for 15 min were immunostained with anti - TOM20 ( red ) anti - DRP1 ( green ) ; arrowheads in far - right panel indicate recruitment of DRP1 onto mitochondrial surface ( TOM20 ) . B , of DRP1 signal on TOM20 positive structures . Errors bars show standard error of mean ( ) ( n = 45 , technical replicates ) .

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A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20). B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates). C


C , Western blot of DRP1 , GAPDH ( marker ) TOM20 ( mitochondrial marker ) in mitochondrial fractions treated or not , with 2µM Mito - C as indicated . D , of western blots showed in C expressed as a distribution of DRP1 in mitochondrial fractions . Errors bars show standard deviation ( SD ) of 5 independent experiments .

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C, Western blot analysis of DRP1, GAPDH (cytosolic marker) and TOM20 (mitochondrial marker) protein in cytosolic and mitochondrial fractions treated or not, with 2µM Mito-C as indicated. D, Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions. Errors bars show the standard deviation (SD) of 5 independent experiments. E


E , HeLa were transfected with DRP1K38A mutant mCherry treated or not , with Mito - C ( T for transfected , NT for not transfected ) . F , of mitochondrial observed in E . Errors bars show standard error of mean ( ) . ( 30 - 35 each with an average of 15 - 20 from triplicate independent experiments were analyzed ) .

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E, HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not, with Mito-C (T for transfected, NT for not transfected). F, Quantification of the mitochondrial phenotypes observed in E. Errors bars show the standard error of the mean (SEM). (30-35 images each with an average of 15-20 cells from triplicate independent experiments were analyzed). G


G , Electron ( EM ) from HeLa treated with 2µM Mito - C or DMSO for 120 minutes . Empty arrowheads indicate ER - mitochondria contact sites . H , of ER - mitochondria contact sites events ( reported in 50µm² section ; 50 from an average of 30 from triplicate independent experiments were analyzed ) . Errors bars show standard error of mean ( ) . I , of ER - mitochondria contact sites density ( in µm ; 30 from an average of 30 from triplicate independent experiments were analyzed ) . Errors bars show standard error of mean ( ) .

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G, Electron micrographs (EM) from HeLa cells treated with 2µM Mito-C or DMSO for 120 minutes. Empty arrowheads indicate ER-mitochondria contact sites. H, Quantification of ER-mitochondria membrane contact sites events (reported in 50µm² section; 50 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). I, Quantification of ER-mitochondria membrane contact sites density (in µm; 30 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM). Da


B , Huh7 were treated with or without ( DMSO ) Mito - C , infected with dengue fixed after 48h of infection . were then immunostained for endogenous TOM20 ( green channel ) , viral non - structural NS5 ( red ) DAPI ( Blue ) . Cropped areas illustrate mitochondrial in described conditions .

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B, Huh7 cells were treated with or without (DMSO) Mito-C, infected with dengue virus and fixed after 48h of infection. Cells were then immunostained for endogenous TOM20 (green channel), viral non-structural protein NS5 (red) and DAPI (Blue). Cropped areas illustrate the mitochondrial morphology in described conditions.


C , of mitochondrial observed in b . Errors bars show standard error of mean ( ) . ( 15 - 20 each with an average of 20 - 25 from triplicate independent experiments were analyzed ) .

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C, Quantification of the mitochondrial phenotypes observed in b. Errors bars show the standard error of the mean (SEM). (15-20 images each with an average of 20-25 cells from triplicate independent experiments were analyzed).


( B ) Gal4 - binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins ( murine ) in Y2H .

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(B) Gal4 DNA-binding domain fusions of MUPs were tested with Gal4 activation domain fusions of ubiquilins (murine) in Y2H.


( C ) HEK293 transfected with indicated MUP - V5 , myc - UBQLN1 ( human ) , or empty vector were immunoprecipitated ( IP ) with anti - myc or anti - UBQLN1 antibodies . Western blot ( WB ) of IP lysate were probed as indicated .

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(C) HEK293 cells transfected with indicated MUP-V5, myc-UBQLN1 (human), or empty vector were immunoprecipitated (IP) with anti-myc or anti-UBQLN1 antibodies. Western blot (WB) of IP and lysate were probed as indicated.


( D - E ) WB of input lysate IP from HEK293 expressing myc - UBQLN1 Rv1926 - V5 ( D ) or Rv1566 - V5 ( E ) . IP was performed using antibodies directed against myc , V5 , or isotype control ( IgG ) .

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(D-E) WB of input lysate and IP from HEK293 cells expressing myc-UBQLN1 and Rv1926-V5 (D) or Rv1566-V5 (E). IP was performed using antibodies directed against myc, V5, or isotype control (IgG).


( B ) Mtb were incubated with lysate from HEK293 transfected with vector control , myc - UBQLN1 , GFP , or V5 - parkin . WB of input ( I ) , 5th wash ( W5 ) , elution ( E ) , probed with indicated antibodies . Actin is a loading control for lysate Ag85b for pellet .

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(B) Mtb were incubated with lysate from HEK293 cells transfected with vector control, myc-UBQLN1, GFP, or V5-parkin. WB analysis of the input (I), 5th wash (W5), and elution (E), probed with indicated antibodies. Actin is a loading control for the cell lysate and Ag85b for the bacterial pellet.


( C ) Mtb were incubated with lysate from HEK293 transfected with myc - UBQLN1 - ΔUBA , myc - UBQLN1 - ΔUBL , myc - UBQLN1 - UBA . WB containing input , 5th wash , elution were incubated with anti - myc antibody . predicted sizes of myc - UBQLN1 - ΔUBL , myc - UBQLN1 - ΔUBA , myc - UBQLN1 - UBA are 55 kD , 50 kD , 6 kD , respectively .

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(C) Mtb were incubated with lysate from HEK293 cells transfected with myc-UBQLN1-ΔUBA, myc-UBQLN1-ΔUBL, and myc-UBQLN1-UBA. WB containing the input, 5th wash, and elution were incubated with anti-myc antibody. The predicted sizes of myc-UBQLN1-ΔUBL, myc-UBQLN1-ΔUBA, and myc-UBQLN1-UBA are 55 kD, 50 kD, and 6 kD, respectively.


( D ) Mtb were incubated with recombinant GST - UBQLN1 , GST - UBQLN1 - ΔUBL , or GST - UBQLN1 - ΔUBA . WB containing input elution was probed with an anti - GST antibody .

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(D) Mtb were incubated with recombinant GST-UBQLN1, GST-UBQLN1-ΔUBL, or GST-UBQLN1-ΔUBA. WB containing the input and elution was probed with an anti-GST antibody.


( A ) BMDMs were infected with GFP - expressing wt Mtb or ΔesxA for 24h immunostained with UBQLN1 antibody . IFN - γ was added 1d prior to infection as indicated . ( B ) of results from A from 3 independent experiments with at least 100 analyzed per experiment ; * * * P < 0 . 001 , Fisher ' s exact , comparing proportion of UBQLN1 positive versus negative Mtb in indicated samples .

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(A) BMDMs were infected with GFP-expressing wt Mtb or ΔesxA for 24h and immunostained with UBQLN1 antibody. IFN-γ was added 1d prior to infection as indicated. (B) Quantification of results from A from 3 independent experiments with at least 100 bacteria analyzed per experiment; ***P< 0.001, Fisher's exact test, comparing the proportion of UBQLN1 positive versus negative Mtb in indicated samples.


( C ) lysate from BMDMs that were treated with IFN - γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 UBQLN2 .

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(C) Cellular lysate from BMDMs that were treated with IFN-γ as indicated were analyzed by WB with an antibody that recognizes UBQLN1 and UBQLN2.


( D ) BMDMs were transfected with siRNAs targeting UBQLN1 or a non - targeting control ( siCON ) lysates were analyzed by WB using an antibody that recognizes UBQLN1 UBQLN2 . ( C - D ) Actin served as a loading control .

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(D) BMDMs were transfected with siRNAs targeting UBQLN1 or a non-targeting control (siCON) and lysates were analyzed by WB using an antibody that recognizes UBQLN1 and UBQLN2. (C-D) Actin served as a loading control.


( E ) BMDMs were infected with Mtb live / dead treated with AnTc 24h prior to fixation .

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(E) BMDMs were infected with Mtb live/dead strain and treated with AnTc 24h prior to fixation.


( F ) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON infected with live / dead Mtb . IFN - γ was added 1d prior to infection if indicated . % Viable were quantified 24 48 hpi . Data are from 2 independent experiments . * P < 0 . 05 ; * * * P < 0 . 001 , Fisher ' s exact , comparing proportion of viable non - viable in indicated samples .

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(F) BMDMs were transfected with siRNAs targeting UBQLN1 or siCON and infected with live/dead Mtb strain. IFN-γ was added 1d prior to infection if indicated. %Viable bacteria were quantified 24 and 48 hpi. Data are from 2 independent experiments. *P<0.05; ***P<0.001, Fisher's exact test, comparing the proportion of viable and non-viable bacteria in indicated samples.


( G ) BMDMs were treated with indicated siRNAs three days prior to infection then activated with IFN - γ one day prior to infection with H37Rv or ΔesxA . CFU were enumerated 4 96 hpi . * P < 0 . 05 , unpaired Student ' s t - . Data are representative of three independent experiments .

O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O 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 were treated with the indicated siRNAs three days prior to infection and then activated with IFN-γ one day prior to infection with H37Rv or ΔesxA. CFU were enumerated 4 and 96 hpi. *P< 0.05, unpaired Student's t-test. Data are representative of three independent experiments.


( H ) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb ( H37Rv ) IFN - γ was measured by

O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O

(H) BMDMs transfected with siRNA targeting UBQLN1 or siCON were infected with wt Mtb (H37Rv) and IFN-γ was measured by ELISA


( I ) UBQLN1 - silenced BMDMs controls were infected with H37Rv or ΔesxA . ( H - I ) 24 hpi infected BMDMs were co - cultured with P25TCR - Tg CD4 + T , IFN - γ was measured by ELISA . Data is representative of 3 independent experiments . * P < 0 . 05 , * * P < 0 . 005 , unpaired Student ' s t - . ( B , F , Γ , H ) Results are mean + / - ; ns , not significant .

O O O B-GENEPROD O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UBQLN1-silenced BMDMs and controls were infected with H37Rv or ΔesxA. (H-I) 24 hpi infected BMDMs were co-cultured with P25TCR-Tg CD4+ T cells, and IFN-γ was measured by ELISA. Data is representative of 3 independent experiments. *P<0.05, **P<0.005, unpaired Student's t-test. (B, F, Γ, H) Results are mean +/- SEM; ns, not significant.


( A ) BMDMs treated with IFN - γ infected for 24h with GFP - Mtb were immunostained for UBQLN1 either ubiquitin ( using FK2 antibody ) or p62 . GFP - LC3 BMDMs infected with DsRed - Mtb were immunostained for UBQLN1 . ( B ) of those Mtb that co - localize with at least one of markers shown in A . More than 375 total were counted in two independent experiments , number co - localizing with at least one marker is indicated as " total " under each bar .

O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O 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) BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and either ubiquitin (using the FK2 antibody) or p62. GFP-LC3 BMDMs infected with DsRed-Mtb were immunostained for UBQLN1. (B) Quantification of those Mtb that co-localize with at least one of the cellular markers shown in A. More than 375 total bacteria were counted in two independent experiments, and the number co-localizing with at least one marker is indicated as "total" under each bar.


( C ) Wt macrophages ( Atg16L1flox / flox ; Cre - ) autophagy - deficient macrophages ( Atg16L1flox / flox Lyz - cre ; Cre + ) were activated with IFN - γ or not , followed by infection with live / dead Mtb reporter . % viable was quantified after addition of AnTc . * * P < 0 . 005 , * * * P < 0 . 0001 , Fisher ' s exact ( in which proportions of viable to non - viable Mtb was compared ) .

O O O O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O 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-ORGANISM O O O O

(C) Wt macrophages (Atg16L1flox/flox; Cre-) and autophagy-deficient macrophages (Atg16L1flox/flox Lyz-cre; Cre+) were activated with IFN-γ or not, followed by infection with the live/dead Mtb reporter strain. % viable was quantified after addition of AnTc. **P<0.005, ***P<0.0001, Fisher's exact test (in which the proportions of viable to non-viable Mtb was compared).


( D ) Atg16L1flox / flox Cre + Cre - BMDMs treated with IFN - γ infected for 24h with GFP - Mtb were immunostained for UBQLN1 p62 . number of Mtb co - localizating with markers is shown . ( C - D ) At least 100 were analyzed in three independent experiments .

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(D) Atg16L1flox/flox Cre+ and Cre- BMDMs treated with IFN-γ and infected for 24h with GFP-Mtb were immunostained for UBQLN1 and p62. The number of Mtb co-localizating with the cellular markers is shown. (C-D) At least 100 bacteria were analyzed in three independent experiments.


( E ) from IFN - γ treated BMDMs of indicated genotype infected with GFP - Mtb immunostained with UBQLN1 p62 . Arrows indicates p62 + UBQLN1 + Mtb .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O B-ORGANISM B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O

(E) Images from IFN-γ treated BMDMs of indicated genotype infected with GFP-Mtb and immunostained with UBQLN1 and p62. Arrows indicates p62+ UBQLN1+ Mtb.


( A - C ) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA . show IFN - γ treated macrophages infected with GFP - Mtb ( A - B ) or DsRed - Mtb ( C ) for 24h . were immunostained for ubiquitin ( Ub ) with FK2 antibody ( A ) or p62 ( B ) , which are shown in red . ( C ) GFP - LC3 BMDMs were infected with DsRed - Mtb . LC3 is shown in green , whereas Mtb are red . ( A - C ) shows of Mtb that colocalized with indicated marker in macrophages that were treated with IFN - γ as indicated . Results are mean + / - from two independent experiments , with at least 100 analyzed per experiment . * * * P < 0 . 001 , Fisher ' s exact ( in which proportion of Mtb associated with marker in a given condition was compared to proportion found in sample treated with siCON IFN - γ ) .

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(A-C) BMDMs transfected with siRNA targeting UBQLN1 or control siRNA. Images show IFN-γ treated macrophages infected with GFP-Mtb (A-B) or DsRed-Mtb (C) for 24h. Cells were immunostained for ubiquitin (Ub) with the FK2 antibody (A) or p62 (B), which are shown in red. (C) GFP-LC3 BMDMs were infected with DsRed-Mtb. LC3 is shown in green, whereas Mtb are red. (A-C) Quantification shows the percentage of Mtb that colocalized with the indicated cellular marker in macrophages that were treated with IFN-γ as indicated. Results are mean +/- SEM from two independent experiments, with at least 100 bacteria analyzed per experiment. ***P<0.001, Fisher's exact test (in which the proportion of Mtb associated with the cellular marker in a given condition was compared to the proportion found in the sample treated with siCON and IFN-γ).


( A ) WT Park2 - / - BMDMs were pretreated with IFN - γ , infected with GFP - Mtb for 24h , immunostained with FK2 antibody . colocalization with FK2 immunoreactivity was quantified from 2 independent experiments .

O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

(A) WT and Park2-/- BMDMs were pretreated with IFN-γ, infected with GFP-Mtb for 24h, and immunostained with the FK2 antibody. Bacterial colocalization with FK2 immunoreactivity was quantified from 2 independent experiments.


( B ) Park2 - / - BMDMs pretreated or not treated with IFN - γ were infected with GFP - Mtb for 24h immunostained with UBQLN1 specific antibody ; Arrows indicate Mtb co - localized with UBQLN1 . ns - not significant .

O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY O B-GENEPROD O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O

(B) Park2-/- BMDMs pretreated or not treated with IFN-γ were infected with GFP-Mtb for 24h and immunostained with UBQLN1 specific antibody; Arrows indicate Mtb co-localized with UBQLN1. ns- not significant.


( C ) Park2 - / - BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection , treated with IFN - γ , infected with GFP - Mtb for 24h prior to immunostaining for ubiquitin ( FK2 antibody ) . Ubiquitin ( FK2 + ) were quantified from two independent experiments . ( A , C ) * * P < 0 . 01 , Fisher ' s exact ( comparing proportion of FK2 positive in indicated samples ) . Results are mean + / - . A minimum of 100 Mtb were counted in each experiment .

O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O

(C) Park2-/- BMDMs were transfected with siRNA targeting UBQLN1 or control siRNA 3d prior to infection, treated with IFN-γ, and infected with GFP-Mtb for 24h prior to immunostaining for ubiquitin (FK2 antibody). Ubiquitin (FK2+) bacteria were quantified from two independent experiments. (A, C) **P<0.01, Fisher's exact test (comparing the proportion of FK2 positive bacteria in indicated samples). Results are mean +/- SEM. A minimum of 100 Mtb were counted in each experiment.


B . Vero E6 were transfected with 10 nM of siRNA before infection by SARS - CoV - 2 at a multiplicity of infection ( MOI ) of 0 . 1 after 24 h . numbers of viral copies were quantified with RT - qPCR . negative control siRNA served as control is abbreviated as " Ctrl . " C1 - C11 represent final candidate sequences after selection . siRNAs capable of inhibiting up to 99 % of viral envelope gene expression with P values < 0 . 005 compared with Ctrl siRNA are marked with * . P value by Student t .

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B. Vero E6 cells were transfected with 10 nM of siRNA before infection by SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 after 24 h. The numbers of viral RNA copies were quantified with RT-qPCR. The negative control siRNA served as the control and is abbreviated as "Ctrl." C1-C11 represent the final candidate sequences after selection. The siRNAs capable of inhibiting up to 99% of viral envelope gene expression with P values < 0.005 compared with Ctrl siRNA are marked with *. P value by Student t test.


E . IC50 of C6 fully modified C6G25S . Vero E6 were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6 or C6G25S before infection at an MOI of 0 . 1 . viral was quantified by RT - qPCR at 24 h after infection . F . IC50 data for viral RdRp inhibition by C6G25S . Vero E6 were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before infection at an MOI of 0 . 1 . viral was quantified by RT - qPCR at 24 h after infection .

O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

E. IC50 of C6 and the fully modified C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6 or C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. F. IC50 data for viral RdRp inhibition by C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. D


B . IC50 for C6G25S against different variants . Vero E6 were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before infection with different strains of SARS - CoV - 2 . viral was quantified by RT - qPCR at 24 h after infection . Data are presented as mean ± SD of three biological replicates .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

B. IC50 for C6G25S against different variants. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before infection with different strains of SARS-CoV-2. The viral RNA was quantified by RT-qPCR at 24 h after infection. Data are presented as mean ± SD of three biological replicates.


A , B , C . K18 - hACE2 - transgenic mice treated with C6G25S by 1 . 48 mg / L of AI for 30 min ( A ) , 50 μL of saline containing 50 μg of C6G25S by IN ( B ) , or PBS ( C ) ( n = 5 per group ) . C6G25S distribution in lungs was visualized by in situ hybridization ( ISH ) with C6G25S - specific probe ( red color ) . Bronchi ( i ) bronchioles ( ii ) marked with boxes are enlarged on right .

O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O

A, B, C. K18-hACE2-transgenic mice treated with C6G25S by 1.48 mg/L of AI for 30 min (A), 50 μL of saline containing 50 μg of C6G25S by IN (B), or PBS (C) (n = 5 per group). C6G25S distribution in lungs was visualized by in situ hybridization (ISH) staining with C6G25S-specific probe (red color). Bronchi (i) and bronchioles (ii) marked with the boxes are enlarged on the right.


E , F . C57 / B6 mice ( n = 3 per group ) were treated by AI with C6G25S 1 . 48 mg / L for 30 min ( E ) or 50 μL of saline containing 50 μg of C6G25S by IN administration ( F ) . C6G25S deposited in whole lungs nasal cavities was quantified after whole tissue homogenization followed by - loop RT - qPCR . data represent mean ± SD . P value by Student t .

O O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

E, F. C57/B6 mice (n = 3 per group) were treated by AI with C6G25S 1.48 mg/L for 30 min (E) or 50 μL of saline containing 50 μg of C6G25S by IN administration (F). The C6G25S deposited in whole lungs and nasal cavities was quantified after whole tissue homogenization followed by stem-loop RT-qPCR. Quantification data represent mean ± SD. P value by Student t test.


A . K18 - hACE2 - transgenic mice ( Winkler et al . , 2020 ) were treated once daily for 3 days before intranasal challenge with 104 plaque - forming units ( PFU ) of original . Prophylactic treatment consists of 30 min of AI ( 1 . 48 mg / l of C6G25S ) , followed by IN of 50 μg C6G25S . Mice receiving vehicle control ( saline ) for both AI IN are annotated as control . Viral ( left ) infectious virions ( right ) in lungs were quantified with RT - qPCR plaque forming , respectively , at 2 days post - infection ( dpi ) . B . Mice were challenged intranasally with 104 PFU of co - treatment with 1 . 48 mg / L of C6G25S or vehicle control ( saline ) by AI for 30 min on day 0 ( right after infection ) day 1 . Viral infectious virions were quantified at 2 dpi .

O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O

A. K18-hACE2-transgenic mice (Winkler et al., 2020) were treated once daily for 3 days before intranasal challenge with 104 plaque-forming units (PFU) of the original virus. Prophylactic treatment consists of 30 min of AI (1.48 mg/l of C6G25S), followed by IN of 50 μg C6G25S. Mice receiving vehicle control (saline) for both AI and IN are annotated as control. Viral RNA (left) and infectious virions (right) in lungs were quantified with RT-qPCR and plaque forming assay, respectively, at 2 days post-infection (dpi). B. Mice were challenged intranasally with 104 PFU of virus and co-treatment with 1.48 mg/L of C6G25S or vehicle control (saline) by AI for 30 min on day 0 (right after infection) and day 1. Viral RNA and infectious virions were quantified at 2 dpi. C


A . Immunohistochemical ( ) of viral spike in lung sections from K18 - hACE2 mice ( Winkler et al . , 2020 ) at day 5 post - infection . Spike were detected with anti - spike antibody stained brown . of bronchial epithelium of vehicle control treated ( i ii ) C6G25S treated ( iii iv ) , alveoli of vehicle control treated ( v vi ) C6G25S treated ( vii viii ) , bronchiole blood vessel of vehicle control treated ( ix x ) C6G25S treated ( xi xii ) group were shown . Syncytial was indicated by green arrow in ( xi ) thrombosis in ( x ) .

O O B-EXP_ASSAY O O O O B-GENEPROD O B-TISSUE O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-DISEASE O O O O O

A. Immunohistochemical (IHC) staining of viral spike proteins in lung sections from K18-hACE2 mice (Winkler et al., 2020) at day 5 post-infection. Spike proteins were detected with anti-spike antibody and stained brown. Images of bronchial epithelium of vehicle control treated (i and ii) and C6G25S treated (iii and iv), alveoli of vehicle control treated (v and vi) and C6G25S treated (vii and viii), bronchiole and blood vessel of vehicle control treated (ix and x) and C6G25S treated (xi and xii) group were shown. Syncytial cell was indicated by green arrow in (xi) and thrombosis in (x).


B . ISH of viral in lungs of vehicle control group ( left ) C6G25S treated group ( right ) at day 2 post - infection . Viral was stained brown as green arrows indicated . are representative of 5 for each group .

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B. ISH staining of viral RNA in lungs of vehicle control group (left) and C6G25S treated group (right) at day 2 post-infection. Viral RNA was stained brown as green arrows indicated. Images are representative of 5 animals for each group.


C . of of Ly6G + ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) C6G25S treated group ( right ) at day 2 post - infection .

O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O

C. Images of IHC staining of Ly6G+ Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection.


D . of of F4 / 80 + ( Brown color ) in lungs of vehicle control ( left ) C6G25S treated group ( right ) at day 2 post - infection . E . of of CD3 + T ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) C6G25S treated group ( right ) at day 2 post - infection .

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D. Images of IHC staining of F4/80+ Cells (Brown color) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. E. Images of IHC staining of CD3 + T Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. F


Representative WBs of RPE - 1 wild - type ( WT ) two selected KO clones for AKAP450 ( akap KO ) , PCNT ( pcnt KO ) CDK5Rap2 ( c5rap2 KO ) probed with indicated antibodies . GM130 was used as a loading

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Representative WBs of RPE-1 wild-type (WT) cells and the two selected KO clones for AKAP450 (akap KO), PCNT (pcnt KO) and CDK5Rap2 (c5rap2 KO) probed with the indicated antibodies. GM130 was used as a loading control


Confocal IF of RPE - 1 WT , single KO ( akap KO , pcnt KO c5rap2 KO ) double KO ( pc - ak - 2KO , left panels ; pc - c5 - 2KO , right panels ) stained with indicated antibodies . yellow dashed indicates nucleus contour ( N ) arrows point centrosome ( CTR ) . GA marks Golgi Apparatus . Scale bars : 5 μm .

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Confocal IF images of RPE-1 WT, single KO (akap KO, pcnt KO and c5rap2 KO) and double KO (pc-ak-2KO, left panels; pc-c5-2KO, right panels) cells stained with the indicated antibodies. The yellow dashed line indicates the nucleus contour (N) and the arrows point the centrosome (CTR). GA marks the Golgi Apparatus. Scale bars: 5 μm.


Representative WB of RPE - 1 WT two selected PCNT / AKAP450 ( pc - ak - 2KO ) PCNT / CDK5Rap2 ( pc - c5 - 2KO ) double knockout mutated clones probed with indicated antibodies . GM130 was used as a loading

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Representative WB of RPE-1 WT cells and the two selected PCNT/AKAP450 (pc-ak-2KO) and PCNT/CDK5Rap2 (pc-c5-2KO) double knockout mutated clones probed with the indicated antibodies. GM130 was used as a loading control


Representative WB of RPE - 1 WT indicated single or double KO lines probed with anti - AKAP450 , PCNT CDK5Rap2 antibodies ( left ) . of signal from three independent WB experiments is shown at right . # indicates KO . arrow points dramatic decrease of CDK5Rap2 in pc - ak - 2KO . Hsp70 was used as a loading

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Representative WB of RPE-1 WT cells and the indicated single or double KO cell lines probed with anti-AKAP450, PCNT and CDK5Rap2 antibodies (left). Quantification of signal intensity from three independent WB experiments is shown at right. # indicates KO proteins. The arrow points the dramatic decrease of CDK5Rap2 levels in pc-ak-2KO cell line. Hsp70 was used as a loading control


Confocal of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO pc - c5 - 2KO stained for EB1 ( green ) centrosomal CAP350 ( red ) . High magnification single - channel of selected areas are shown at right . Scatter plot showing of EB1 comets from three independent experiments as that shown in A . A region of interest of 3 μm radius around CAP350 signal was used to number of comets . Horizontal black lines represent mean .

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Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) and the centrosomal protein CAP350 (red). High magnification single-channel images of selected areas are shown at right. Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A. A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets. Horizontal black lines represent the mean.


MT regrowth experiments of WT mixed with either WT ( as a control ) , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO or pc - c5 - 2KO . Samples were fixed 3 min after NZ washout stained for EB1 ( green ) indicated in red channel . of EB1 signal around

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MT regrowth experiments of WT cells mixed with either WT (as a control), akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO or pc-c5-2KO cells. Samples were fixed 3 min after NZ washout and stained for EB1 (green) and the indicated protein in the red channel. Quantification of EB1 signal intensity around the centrosome


WT pc - ak - 2KO were transfected with a combination of three siRNAs against CEP192 , fixed triple labelled for EB1 , CEP192 CAP350 , as a centrosomal marker . Scramble siRNA was used as a control .

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WT and pc-ak-2KO cells were transfected with a combination of three siRNAs against CEP192, fixed and triple labelled for EB1, CEP192 and CAP350, as a centrosomal marker. Scramble siRNA was used as a control.


Confocal of WT , akap KO , c5rap2 KO , pcnt KO , pc - ak - 2KO pc - c5 - 2KO stained for EB1 ( green ) EB1 comets around

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Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) EB1 comets around the centrosome


Three min MT regrowth experiments in WT pc - ak - 2KO transfected with either scramble or CEP192 siRNAs .

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Three min MT regrowth experiments in WT and pc-ak-2KO cells transfected with either scramble or CEP192 siRNAs.


Quantification of the EB1 signal intensity around the centrosome


MT regrowth experiments in WT c5rap2 KO or pcnt KO . were stained as indicated . Arrows point centrosome .

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MT regrowth experiments in WT and c5rap2 KO or pcnt KO cells. Cells were stained as indicated. Arrows point the centrosome.


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Quantification of EB1 signal intensity at Golgi membranes.


WB of AKAP450 co - immunoprecipitation assays revealed with indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant .

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WB of AKAP450 co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.


WB of AKAP450 PCNT ( B ) co - immunoprecipitation assays revealed with indicated antibodies . Asterisks mark immunoglobulin heavy chains . Cntrl : non - specific Igs immunoprecipitates ; IP : specific Igs immunoprecipitates ; SP : supernatant .

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WB of AKAP450 and PCNT (B) co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.


Centrosomes from WT , single double KO , as indicated , were labelled for AKAP450 PCNT Right panels show quantifications . A region of interest of 1 , 5 μm radius was drawn around centrosome was determined normalized to WT mean .

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Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.