Figure 3A Localization on the lnc - SMaRT transcript of the two sets of biotinylated probes ( Set # 1 and Set # 2 ) used for lnc - SMaRT pull - down experiment . B Left panel : qPCR analysis of lnc - SMaRT enrichment in the RNA pull - down performed in C2C12 cells at day 2 of differentiation ( DM2 ) ; Set # 1 and Set # 2 probes were used against lnc - SMaRT together with a control set of probes against LacZ mRNA ( LacZ ) . Data are expressed in percentage of input and presented as the mean ± s . e . m . of three biological replicates ( dots ) . Right panel : Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc - SMaRT pull - down ; GAPDH was used as the negative control . Representative results from three independent experiments are shown . C Upper panel : Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation . Input sample ( IN ) accounts for 2 . 5 % of the extract . Representative results from three independent experiments are shown . Lower panel : qPCR analysis of lnc - SMaRT enrichment in DHX36 RIP - derived RNA extracts . WBP4 was used as positive control [ 31 ] , while Neat1 ( a lncRNA expressed at comparable level of lnc - SMaRT according to the RNA - sequencing ) and Rps7 were used as negative controls . Data are expessed as percentage of input normalized on IgG control , and presented as the mean ± s . e . m . of three biological replicates ( dots ) . D RT - PCR validation of Mlx - α , Mlx - β and MLX - γ mRNA enrichment upon the lnc - SMaRT pull - down performed with Set # 1 and Set # 2 probes ; a control set of probes against LacZ mRNA ( LacZ ) was used as negative control . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown . E RT - PCR analysis of Mlx - α , Mlx - β and Mlx - γ mRNA enrichment in DHX36 RIP - derived RNA extracts in samples treated with control siRNA ( siSCR , upper panel ) or siRNA against lnc - SMaRT ( siSMaRT , lower panel ) . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown . F Upper panel : Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG - tagged MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . FLAG - tag has been inserted at the N - terminus of MLX protein isoforms . HPRT was used as loading control . Representative results from three independent experiments are shown . Middle panel : Quantification of FLAG - tagged protein levels normalized on HPRT signals . Data are expressed presented as the mean ± s . d . of three biological replicates ( dots ) . Statistical analysis was performed with two - way ANOVA followed by Bonferroni ' s multiple comparisons test . * * * P < 0 . 001 . Lower panel : RT - PCR analysis of lnc - SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged - MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . GAPDH was used as loading control . Representative results from three independent experiments are shown . G Upper panel : Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector ( CTRL ) or with a plasmid overexpressing FLAG - tagged MLX - γ ( MLX - γ ) in control conditions ( si - SCR ) or in knockdown of DHX36 ( si - DHX36 ) . ACTININ was used as loading control . Representative results from three independent experiments are shown . Lower panel : RT - PCR analysis of Mlx - γ expression on the described RNA extracts . GAPDH was used as loading control . Representative results from three independent experiments are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Localization on the lnc-SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc-SMaRT pull-down experiment.B Left panel: qPCR analysis of lnc-SMaRT enrichment in the RNA pull-down performed in C2C12 cells at day 2 of differentiation (DM2); Set#1 and Set#2 probes were used against lnc-SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots). Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc-SMaRT pull-down; GAPDH was used as the negative control. Representative results from three independent experiments are shown. C Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown. Lower panel: qPCR analysis of lnc-SMaRT enrichment in DHX36 RIP-derived RNA extracts. WBP4 was used as positive control [31], while Neat1 (a lncRNA expressed at comparable level of lnc-SMaRT according to the RNA-sequencing) and Rps7 were used as negative controls. Data are expessed as percentage of input normalized on IgG control, and presented as the mean ± s.e.m. of three biological replicates (dots). D RT-PCR validation of Mlx-α, Mlx-β and MLX-γ mRNA enrichment upon the lnc-SMaRT pull-down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.E RT-PCR analysis of Mlx-α, Mlx-β and Mlx-γ mRNA enrichment in DHX36 RIP-derived RNA extracts in samples treated with control siRNA (siSCR, upper panel) or siRNA against lnc-SMaRT (siSMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.F Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG-tagged MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). FLAG-tag has been inserted at the N-terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown. Middle panel: Quantification of FLAG-tagged protein levels normalized on HPRT signals. Data are expressed presented as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two-way ANOVA followed by Bonferroni's multiple comparisons test. ***P < 0.001. Lower panel: RT-PCR analysis of lnc-SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged-MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). GAPDH was used as loading control. Representative results from three independent experiments are shown. G Upper panel: Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector (CTRL) or with a plasmid overexpressing FLAG-tagged MLX-γ (MLX-γ) in control conditions (si-SCR) or in knockdown of DHX36 (si-DHX36). ACTININ was used as loading control. Representative results from three independent experiments are shown. Lower panel: RT-PCR analysis of Mlx-γ expression on the described RNA extracts. GAPDH was used as loading control. Representative results from three independent experiments are shown.


Figure 4A Immunofluorescence for total MLX protein detection ( red signal ) performed on C2C12 cells treated with siRNAs against Mlx - γ ( si - Mlx - γ ) , lnc - SMaRT ( si - SMaRT ) , DHX36 ( si - DHX36 ) or with a control siRNA ( si - SCR ) . Cells were fixed two days after the switch to differentiation medium . Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment . The inserts on the topside of the images sketch the MLX signal peaks in the nuclei ( white stars ) to highlight the variation of MLX staining in each condition . Dashed lines indicate the edge of the nucleus . B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear ( Nuc ) and cytoplasmic ( Cyt ) compartment in the indicated conditions . About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates ( dots ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Immunofluorescence for total MLX protein detection (red signal) performed on C2C12 cells treated with siRNAs against Mlx-γ (si-Mlx-γ), lnc-SMaRT (si-SMaRT), DHX36 (si-DHX36) or with a control siRNA (si-SCR). Cells were fixed two days after the switch to differentiation medium. Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment. The inserts on the topside of the images sketch the MLX signal peaks in the nuclei (white stars) to highlight the variation of MLX staining in each condition. Dashed lines indicate the edge of the nucleus.B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear (Nuc) and cytoplasmic (Cyt) compartment in the indicated conditions. About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates (dots).


Figure 5A Left panel : schematic representation of the luciferase constructs produced for Mlx . Mlx 5 ' - and 3 ' UTR - interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region ( RLuc - Mlx 5 ' - 3 ' ) and mutant derivatives devoid of the 5 ' interacting region ( RLuc - Mlx - 3 ' ) or the 3 ' UTR interacting region ( RLuc - Mlx - 5 ' ) were obtained . These constructs were co - transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx 5 ' - 3 ' , RLuc - Mlx - 3 ' , RLuc - Mlx - 5 ' ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * P < 0 . 05 . B The RLuc - Mlx - 5 ' construct was co - transfected in N2a cells together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) and treated with control siRNA ( + ) or siRNA against DHX36 ( - ) . Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to RLuc - Mlx - 5 ' vector set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * P < 0 . 01 , * * * P < 0 . 001 . C Left panel : schematic representation of the luciferase constructs used . The RLuc - Mlx - 5 ' construct and its derived mutants ( RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) were transfected in N2a cells in growth conditions together with lnc - SMaRT - expressing plasmids ( lnc - SMaRT ) or with its derived mutant ( lnc - SMaRTmut ) in the indicated combinations . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx - 5 ' , RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * * P < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Left panel: schematic representation of the luciferase constructs produced for Mlx. Mlx 5'- and 3'UTR-interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region (RLuc-Mlx 5'-3') and mutant derivatives devoid of the 5' interacting region (RLuc-Mlx-3') or the 3' UTR interacting region (RLuc-Mlx-5') were obtained. These constructs were co-transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc-SMaRT (+) or with a control vector (-). Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx 5'-3', RLuc-Mlx-3', RLuc-Mlx-5') set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P < 0.05.B The RLuc-Mlx-5' construct was co-transfected in N2a cells together with plasmids expressing lnc-SMaRT (+) or with a control vector (-) and treated with control siRNA (+) or siRNA against DHX36 (-). Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to RLuc-Mlx-5' vector set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P < 0.01, ***P < 0.001.C Left panel: schematic representation of the luciferase constructs used. The RLuc-Mlx-5' construct and its derived mutants (RLuc-Mlxmut30, RLuc-Mlx Δ75) were transfected in N2a cells in growth conditions together with lnc-SMaRT-expressing plasmids (lnc-SMaRT) or with its derived mutant (lnc-SMaRTmut) in the indicated combinations. Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx-5', RLuc-Mlxmut30, RLuc-Mlx Δ75) set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


Figure 6A Upper panel : schematic representation of the predicted base pairing between lnc - SMaRT ( region A ) and Mlx γ mRNA ; the predicted G - 4 structure is boxed in red while the synthetic oligos sequences are underlined in red . Lower panel : Lane 1 and 2 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA ( SYBR GOLD , indicated as SYBR in the figure ) . Lane 3 and 4 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 5 and 6 : 200 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX ( NMM ) . Lane 7 and 8 : 5 pmol of Mlx oligo alone ( 7 ) or in combination with 5pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 9 and 10 : 200 pmol of Mlx oligo alone ( 7 ) or in combination with 200 pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX . Asterisk ( * ) indicates the second NMM - positive band obtained upon SMaRT and γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown . B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel . Oligos were treated as described . ° C = oligos were heated ( + ) or were not heated ( - ) at 100 ° C for 10 min and loaded after slow cooling . KCl = oligos were incubated ( + ) or were not ( - ) in a buffer containing 100mM KCl ( for G - quadruplex stabilization ) . The gel was stained with total RNA staining ( SYBR GOLD ) . Data information : Representative gels from at least three independent experiments are shown . C 200 pmol of indicated oligos treated as in ( B ) were loaded on a native polyacrylamide gel with the selective G - quadruplex staining N - methyl mesoporphyrin IX . Asterisk ( * ) indicates the second NMM - positive band obtained upon SMaRT and γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.B 5 pmol of indicated oligos were loaded on a native polyacrylamide gel. Oligos were treated as described. °C = oligos were heated (+) or were not heated (-) at 100 °C for 10 min and loaded after slow cooling. KCl= oligos were incubated (+) or were not (-) in a buffer containing 100mM KCl (for G-quadruplex stabilization). The gel was stained with total RNA staining (SYBR GOLD). Data information: Representative gels from at least three independent experiments are shown.C 200 pmol of indicated oligos treated as in (B) were loaded on a native polyacrylamide gel with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.


Figure 2C ) ykt6ts pho8 ∆ 60 cells containing an empty plasmid , 3HA - Ykt6 or 3HA - tagged Ykt6 mutant variants as indicated were analyzed as in A ) . D ) ykt6ts pho8 ∆ 60 cells containing an empty plasmid , 3HA - Ykt6 , or ADH1 promoter driven 3HA - Ykt6 wild type or 3HA - tagged Ykt6 mutant variants as indicated were grown and Pho8 ∆ 60 activity was monitored as described in A ) . E ) Ykt6 levels were analyzed from the strains shown in D ) and corresponding cell extracts were analyzed by anti - HA and anti - Pgk1 Western blotting .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2C) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6 or 3HA-tagged Ykt6 mutant variants as indicated were analyzed as in A).D) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6, or ADH1 promoter driven 3HA-Ykt6 wild type or 3HA-tagged Ykt6 mutant variants as indicated were grown and Pho8∆60 activity was monitored as described in A).E) Ykt6 levels were analyzed from the strains shown in D) and corresponding cell extracts were analyzed by anti-HA and anti-Pgk1 Western blotting.


Figure 3A ) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC . Cells were lysed and the total extract ( T ) separated into a cytoplasmic ( S ) and a 100 ' 000x g membrane pellet ( P ) fraction . Fractions were analyzed by anti - HA , anti - Tom70 and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown . B ) vam3 ∆ or ykt6ts vam3 ∆ cells containing the indicated centromeric plasmids were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ° C for 1 hour followed by 1 hour starvation in SD - N at 37 ºC . After cell lysis , samples were subjected to proteinase K ( ProtK ) and Triton X - 100 ( TX100 ) treatment as indicated and analyzed by anti - Ape1 Western blotting . Ape1 * : proteinase K - resistant fragment of Ape1 . One representative experiment out of three biological replicates is shown . C ) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid , 3HA - Ykt6 wild type , or 3HA - tagged Ykt6 mutant variants . Cells were grown at the permissive temperature ( 23 ºC ) to late exponential phase in selective medium , shifted to the restrictive temperature ( 37 ° C ) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting . Samples were analyzed by anti - CPY and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown . D ) Exponentially growing ykt6ts cells at permissive temperature ( 23 ºC ) containing centromeric plasmids as indicated were shifted to the restrictive temperature ( 37 ºC ) for 1 hour before starvation for 4 hours in SD - N . Trichloroacetic acid extracts were prepared . Lipidation of Atg8 was analyzed on a 15 % SDS - PAGE containing 6 M urea by anti - Atg8 and anti - Pgk1 Western blotting . One representative experiment out of three biological replicates is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC. Cells were lysed and the total extract (T) separated into a cytoplasmic (S) and a 100'000x g membrane pellet (P) fraction. Fractions were analyzed by anti-HA, anti-Tom70 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.B) vam3∆ or ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown.C) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.


Figure 4A and B ) GFP - Atg8 atg1 ∆ , GFP - Atg8 atg1 ∆ vam3 ∆ , GFP - Atg8 wild type , GFP - Atg8 vam3 ∆ , GFP - Atg8 ykt6ts or GFP - Atg8 ykt6ts vam3 ∆ cell containing centromeric plasmids as indicated were grown at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD - N . Representative microscopy images are shown in A ) . GFP - Atg8 puncta per cell were quantified from 3 biological replicates ( n ≥ 100 cells ) and the mean was plotted in B ) . Error bars are SD . Scale bar : 5 µm . Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ cells containing centromeric plasmids as indicated . Cells were starved for 16 hours at the permissive temperature ( 23 ° C ) to allow formation of mature , closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ cells grown under rich conditions at 30 ° C . Fusion reactions were incubated at the restrictive temperature ( 30 ° C ) for 2 hours . Representative images are shown in C ) . Autophagosomes were prepared from GFP - Atg8 vam3 ∆ pep4 ∆ or GFP - Atg8 ykt6ts vam3 ∆ pep4 ∆ cells containing centromeric plasmids as indicated . Cells were starved for 16 hours at the permissive temperature ( 23 ° C ) to allow formation of mature , closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting . Vacuoles were isolated from Vph1 - 4xmCherry atg15 ∆ pep4 ∆ cells grown under rich conditions at 30 ° C . Fusion reactions were incubated at the restrictive temperature ( 30 ° C ) for 2 hours . The graph in D ) shows the mean from at least three independent biological experiments . Error bars represent the standard deviation . The indicated strains were grown to late exponential phase at permissive temperature ( 23 ºC ) , shifted to the restrictive temperature at 37 ° C for 1 hour followed by starvation in SD - N for 1 hour at 37 ºC . 3HA - Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated ( IP ) and analyzed by anti - HA Western blotting . Co - precipitating proteins were analyzed by anti - Vam3 and anti - Vti1 Western blotting . One representative experiment out of three biological replicates is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A and B) GFP-Atg8 atg1∆, GFP-Atg8 atg1∆ vam3∆, GFP-Atg8 wild type, GFP-Atg8 vam3∆, GFP-Atg8 ykt6ts or GFP-Atg8 ykt6ts vam3∆ cell containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD-N. Representative microscopy images are shown in A). GFP-Atg8 puncta per cell were quantified from 3 biological replicates (n≥100 cells) and the mean was plotted in B). Error bars are SD. Scale bar: 5 µm.Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. Representative images are shown in C).Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. The graph in D) shows the mean from at least three independent biological experiments. Error bars represent the standard deviation.The indicated strains were grown to late exponential phase at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by starvation in SD-N for 1 hour at 37 ºC. 3HA-Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated (IP) and analyzed by anti-HA Western blotting. Co-precipitating proteins were analyzed by anti-Vam3 and anti-Vti1 Western blotting. One representative experiment out of three biological replicates is shown.


Figure 1X - ray analysis of 6 - months - old H2 - c - fosLTR / Egfrwa / + and H2 - c - fosLTR / Egfrwa / wa littermates . Scale bars : 1cmBone tumor number per mouse at 5 - 6 months of age ( n = 23 wa / + , 15 wa / wa ) Quantification of tumor size in tibiae at 5 - 6 months of age ( n = 23 wa / + , 15 wa / wa ) Alkaline Phosphatase ( ALP ) levels in the serum at endpoint ( age = 5 - 6 months ; n = 16 wa / + , 11 wa / wa ) X - ray analysis before ( age = 2 months ) and after ( age = 7 months ) vehicle or Erlotinib treatmentQuantification of tumor number during treatment ( n = 6 Vehicle , 5 Erlotinib ) Tumor size during treatment ( n = 6 Vehicle , 5 Erlotinib ) Analysis of serum ALP levels during treatment ( n = 5 ) PET summation images ( 0 - 90 min ) in horizontal ( upper panel ) and coronal view ( lower panel ) depicting [ 11C ] Erlotinib distribution in one H2 - c - fosLTR / Egfrwt mouse ( M125 ) . Anatomical structures are labelled with arrows ( T , tumor ; L , liver ; H , heart ; B , brain ) . Scale bars : 1cm Concentration - time curves of [ 11C ] Erlotinib in bone tumors in right scapula of three H2 - c - fosLTR / Egfrwt mice measured with PET . Broken line indicates threshold for in vitro effect of Erlotinib ( 1 µM )

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Figure 1X-ray analysis of 6-months-old H2-c-fosLTR/Egfrwa/+ and H2-c-fosLTR/Egfrwa/wa littermates. Scale bars: 1cmBone tumor number per mouse at 5-6 months of age (n=23 wa/+, 15 wa/wa)Quantification of tumor size in tibiae at 5-6 months of age (n=23 wa/+, 15 wa/wa)Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=16 wa/+, 11 wa/wa)X-ray analysis before (age = 2 months) and after (age = 7 months) vehicle or Erlotinib treatmentQuantification of tumor number during treatment (n=6 Vehicle, 5 Erlotinib)Tumor size during treatment (n=6 Vehicle, 5 Erlotinib)Analysis of serum ALP levels during treatment (n=5)PET summation images (0-90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [11C]Erlotinib distribution in one H2-c-fosLTR/Egfrwt mouse (M125). Anatomical structures are labelled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1cm Concentration-time curves of [11C]Erlotinib in bone tumors in right scapula of three H2-c-fosLTR/Egfrwt mice measured with PET. Broken line indicates threshold for in vitro effect of Erlotinib (1 µM)


Figure 2X - ray analysis of 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates . Scale bars : 1cmBone tumor number per mouse at 6 - 7 months of age ( n = 22 wt , 11 ∆ Ob ) Quantification of tumor size in tibiae ( n = 22 wt , 11 ∆ Ob ) ALP levels in the serum at 7 - month endpoint ( n = 17 wt , 8 ∆ Ob ) µPET / CT analysis of 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates Standardized uptake values ( SUV ) of the µPET - tracer Na [ 18F ] F in the pelvic osteosarcoma of 4 - and 7 - months - old H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob mice . n = 6 wt , 4 ∆ Ob for 4 month time - point , n = 6 wt , 3 ∆ Ob for 7 month time -

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Figure 2X-ray analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 1cmBone tumor number per mouse at 6-7 months of age (n=22 wt, 11 ∆Ob) Quantification of tumor size in tibiae (n=22 wt, 11 ∆Ob)ALP levels in the serum at 7-month endpoint (n=17 wt, 8 ∆Ob)µPET/CT analysis of 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates Standardized uptake values (SUV) of the µPET-tracer Na[18F]F in the pelvic osteosarcoma of 4- and 7-months-old H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice. n=6 wt, 4 ∆Ob for 4 month time-point, n=6 wt, 3 ∆Ob for 7 month time-point


Figure 3PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells ( for PCNA ) and as positive cells per mm² ( for cleaved Caspase 3 ) in OS from H2 - c - fosLTR / Egfrwt ( n = 6 ) and H2 - c - fosLTR / Egfr ∆ Ob ( n = 5 ) mice . Scale bars : 100µmEgfr , Ccnd1 , c - fos and transgenic c - fos ( c - fostg ) mRNA expression levels in tumors of H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob mice normalized to Tbp . n = 17 wt , 14 ∆ Ob ( Egfr , Ccnd1 ) , n = 16 wt , 13 ∆ Ob ( c - fos ) , n = 16 wt , 14 ∆ Ob ( c - fostgIHC staining and quantification showing pRSK2 ( n = 4 ) , pCREB ( n = 7 wt , 6 ∆ Ob ) and c - Fos ( n = 5 ) positive cells ( % ) in OS from H2 - c - fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob littermates . Scale bars :

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3PCNA and cleaved Caspase 3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm² (for cleaved Caspase 3) in OS from H2-c-fosLTR/Egfrwt (n=6) and H2-c-fosLTR/Egfr∆Ob (n=5) mice. Scale bars: 100µmEgfr, Ccnd1, c-fos and transgenic c-fos (c-fostg) mRNA expression levels in tumors of H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob mice normalized to Tbp. n=17 wt, 14 ∆Ob (Egfr, Ccnd1), n=16 wt, 13 ∆Ob (c-fos), n=16 wt, 14 ∆Ob (c-fostgIHC staining and quantification showing pRSK2 (n=4), pCREB (n=7 wt, 6∆Ob) and c-Fos (n=5) positive cells (%) in OS from H2-c-fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob littermates. Scale bars: 100µm


Figure 4Western Blot analysis of primary OS cells isolated from H2 ‑ c ‑ fosLTR / Egfrwt and H2 - c - fosLTR / Egfr ∆ Ob micec - fos and transgenic c - fos ( c fostg ) mRNA expression levels in primary H2 - c - fosLTR OS cells after 4x in vitro passages , cultured under standard conditions ( n = 3 independent cell lines ) Western Blot analysis of H2 - c - fosLTR / Egfrwt OS cells treated for 24h with Erlotinibc - fos and c - fostg mRNA expression levels in H2 - c - fosLTR / Egfrwt OS cells treated for 24h with Erlotinib ( 10µM ) or DMSO as control ( n = 4 independent cell lines ) Western Blot analysis of starved H2 - c - fosLTR / Egfrwt OS cells , pre - treated with DMSO ( 1 : 1000 ) , Afatinib ( 5µM ) , GSK2233470 ( 10µM ) , Rapamycin ( 10nM ) or U0126 ( 10µM ) for 30 minutes and stimulated with EGF ( 50ng / ml ) as indicatedWestern Blot analysis of primary OS cells isolated from a p53f / f Rb1f / f Osx - Cre mouse after 24h Erlotinib treatmentc - fos mRNA expression levels in p53f / f Rb1f / f Osx - Cre OS cells treated for 24h with Erlotinib ( 10µM ) ( n = 3 ) Western Blot analysis of starved p53f / f Rb1f / f Osx - Cre OS cells , pre - treated with DMSO ( 1 : 1000 ) or Afatinib ( 5µM ) for 30 minutes and stimulated with EGF ( 50ng / ml ) as

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Western Blot analysis of primary OS cells isolated from H2‑c‑fosLTR/Egfrwt and H2-c-fosLTR/Egfr∆Ob micec-fos and transgenic c-fos (c fostg) mRNA expression levels in primary H2-c-fosLTR OS cells after 4x in vitro passages, cultured under standard conditions (n=3 independent cell lines)Western Blot analysis of H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinibc-fos and c-fostg mRNA expression levels in H2-c-fosLTR/Egfrwt OS cells treated for 24h with Erlotinib (10µM) or DMSO as control (n=4 independent cell lines)Western Blot analysis of starved H2-c-fosLTR/Egfrwt OS cells, pre-treated with DMSO (1:1000), Afatinib (5µM), GSK2233470 (10µM), Rapamycin (10nM) or U0126 (10µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicatedWestern Blot analysis of primary OS cells isolated from a p53f/f Rb1f/f Osx-Cre mouse after 24h Erlotinib treatmentc-fos mRNA expression levels in p53f/f Rb1f/f Osx-Cre OS cells treated for 24h with Erlotinib (10µM) (n=3)Western Blot analysis of starved p53f/f Rb1f/f Osx-Cre OS cells, pre-treated with DMSO (1:1000) or Afatinib (5µM) for 30 minutes and stimulated with EGF (50ng/ml) as indicated


Figure 5X - ray analysis of 6 - months - old H2 - c - fosLTR and H2 - c - fosLTR / ColAREG littermates . Scale bars : 1cmBone tumor number per mouse at 5 - 6 months of age ( n = 22 wt , 13 ColAREG ) Quantification of tumor size in tibiae at 5 - 6 months of age ( n = 22 wt , 13 ColAREG ) Alkaline Phosphatase ( ALP ) levels in the serum at endpoint ( age = 5 - 6 months ; n = 29 wt , 19 ColAREG ) c - fos mRNA expression levels in osteosarcomas of H2 - c - fosLTR ( n = 11 ) and H2 - c - fosLTR / ColAREG mice ( n = 14 ) Western Blot analysis of bone tumor protein lysates from H2 - c - fosLTR and H2 - c - fosLTR / ColAREG

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Figure 5X-ray analysis of 6-months-old H2-c-fosLTR and H2-c-fosLTR/ColAREG littermates. Scale bars: 1cmBone tumor number per mouse at 5-6 months of age (n=22 wt, 13 ColAREG)Quantification of tumor size in tibiae at 5-6 months of age (n=22 wt, 13 ColAREG)Alkaline Phosphatase (ALP) levels in the serum at endpoint (age = 5-6 months; n=29 wt, 19 ColAREG)c-fos mRNA expression levels in osteosarcomas of H2-c-fosLTR (n=11) and H2-c-fosLTR/ColAREG mice (n=14)Western Blot analysis of bone tumor protein lysates from H2-c-fosLTR and H2-c-fosLTR/ColAREG mice


Figure 6Representative images of OS biopsies stained with antibodies against EGFR and c ‑ Fos . Scale bars : 50µmKaplan - Meier survival curve comparing the survival of patients with EGFR and c - Fos double positive osteosarcomas against patients without co - expression of both proteins . ( n = 52 )

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Figure 6Representative images of OS biopsies stained with antibodies against EGFR and c‑Fos. Scale bars: 50µmKaplan-Meier survival curve comparing the survival of patients with EGFR and c-Fos double positive osteosarcomas against patients without co-expression of both proteins. (n=52)


Figure 7Western Blot analysis showing EGFR and c - Fos protein levels in human OS cell lines cultured under standard conditionsCell viability of 143b or LM7 cells cultured for 24h in medium ( + 10 % FCS ) with DMSO ( 1 : 2000 ) or Erlotinib ( 10µM ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to DMSO ) Cell viability of 143b or LM7 cells cultured for 24h in medium ( + 0 . 5 % FCS ) containing 1xPBS ( contr . , 1 : 200 ) or EGF ( 50ng / ml ) ( n = 3 , representative result from 3 independent experiments , shown as fold change , normalized to control ) 143b xenograft growth curve during therapeutic regime ( n = 8 vehicle , 6 Erlotinib ; two independent experiments ) 143b tumor weight at endpoint ( n = 8 Vehicle , 6 Erlotinib ; two independent experiments ) LM7 xenograft growth curve during therapeutic regime ( n = 7 Vehicle , 5 Erlotinib ; two independent experiments ) LM7 tumor weight at endpoint . n = 7 vehicle , 5 Erlotinib , two independent experimentIHC analysis of PCNA and cleaved Caspase 3 in 143b - derived tumors ( n = 4 ) . Scale bars : 50µm IHC analysis of pCREB and c - Fos in 143b - derived tumors . ( n = 5 for pCREB and n = 4 for c - Fos analysis ) . Scale bars : 50µmWestern Blot analysis of pCREB / CREB and p - c - Fos / c - Fos protein expression in lysates directly isolated from 143b xenografts at endpoint , after 14 days of vehicle or Erlotinib

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Western Blot analysis showing EGFR and c-Fos protein levels in human OS cell lines cultured under standard conditionsCell viability of 143b or LM7 cells cultured for 24h in medium (+10% FCS) with DMSO (1:2000) or Erlotinib (10µM) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to DMSO)Cell viability of 143b or LM7 cells cultured for 24h in medium (+0.5% FCS) containing 1xPBS (contr., 1:200) or EGF (50ng/ml) (n=3, representative result from 3 independent experiments, shown as fold change, normalized to control)143b xenograft growth curve during therapeutic regime (n=8 vehicle, 6 Erlotinib; two independent experiments) 143b tumor weight at endpoint (n=8 Vehicle, 6 Erlotinib; two independent experiments)LM7 xenograft growth curve during therapeutic regime (n=7 Vehicle, 5 Erlotinib; two independent experiments) LM7 tumor weight at endpoint. n=7 vehicle, 5 Erlotinib, two independent experimentIHC analysis of PCNA and cleaved Caspase 3 in 143b-derived tumors (n=4). Scale bars: 50µm IHC analysis of pCREB and c-Fos in 143b-derived tumors. (n=5 for pCREB and n=4 for c-Fos analysis). Scale bars: 50µmWestern Blot analysis of pCREB/CREB and p-c-Fos/c-Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or Erlotinib treatment


Figure 1 ( B ) Automatic cluster - mapping of the temperature - dependent splicing changes in CHX , based on changes in PSI ( percent spliced in ) . Categories ( right ) were manually assigned . ( C ) Examples for cold - induced ( blue ) or heat - induced ( red ) AS - decay via exon inclusion or exon skipping . In each example , on top a simplified exon - intron structure is given , PTCs and the length of frameshift inducing exons are highlighted . Predicted splicing changes were validated by radioactive RT - PCR with primers binding to the surrounding constitutive exons . Box plots show median PSI of validation PCRs of four different samples from at least two different mice , whiskers show min to max . Individual data points are shown as circles .

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

Figure 1 (B) Automatic cluster-mapping of the temperature-dependent splicing changes in CHX, based on changes in PSI (percent spliced in). Categories (right) were manually assigned.  (C) Examples for cold-induced (blue) or heat-induced (red) AS-decay via exon inclusion or exon skipping. In each example, on top a simplified exon-intron structure is given, PTCs and the length of frameshift inducing exons are highlighted. Predicted splicing changes were validated by radioactive RT-PCR with primers binding to the surrounding constitutive exons. Box plots show median PSI of validation PCRs of four different samples from at least two different mice, whiskers show min to max. Individual data points are shown as circles.


Figure 2 ( A ) Temperature - dependent poison events in Hnrnpdl ( left , see also Figure 1C ) , Hnrnph3 ( middle ) and Cirbp ( right ) . For each target , on top a simplified exon - intron structure is given and below Sashimi plots show the distribution of raw sequencing reads . Exon - Exon junction reads are indicated by the numbers connecting the exons . Below , sequence conservation across placental species is indicated . Hnrnpdl exhibits a heat - included exon that leads to a PTC , Hnrnph3 a cold - skipped exon that leads to a frameshift , and in Cirbp , heat leads to inclusion of an alternative transcript end , which leads to formation of a PTC . ( B ) For each gene from A , the triplicate normalized read counts in DMSO ( left y - axis , green ) and the percentage of the poison isoform in CHX ( right y - axis , orange ) are plotted at the two temperatures . Line represents mean PSI . ( C ) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature - dependent AS - decay skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform between the CHX samples . Top shows the GE change between the DMSO samples and bottom between the CHX samples . R² and P ( deviation from zero slope ) are indicated . N = 24 . ( E ) Splicing reporters , containing only an intron ( GFP ctrl , # 1 ) or additionally temperature - dependent exons with ~ 1kB surrounding introns . Representative radioactive RT - PCRs ( bottom ) confirm cold - induced inclusion of Ythdf3 exon 7 ( # 2 ) and human Synerg exon 21 ( # 3 ) after 24 hours at the indicated temperatures . Representative images are derived from three independent gels . See SourceDataForFigure2E for uncropped versions of these gels . ( F - G ) GFP expression was quantified using FACS and is shown relative to GFP ctrl . In ( F ) transfected cells were incubated at the indicated temperatures for 24 hours ( n = 3 , mean + / - SD ) . In ( G ) cells were incubated directly after transfection for 12 hours at 39 ° C ( blue ) or 33 ° C ( red ) and then shifted for 2 , 4 , 8 and 12 hours ( n = 3 , mean + / - SD ) .

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

Figure 2 (A) Temperature-dependent poison events in Hnrnpdl (left, see also Figure 1C), Hnrnph3 (middle) and Cirbp (right). For each target, on top a simplified exon-intron structure is given and below Sashimi plots show the distribution of raw sequencing reads. Exon-Exon junction reads are indicated by the numbers connecting the exons. Below, sequence conservation across placental species is indicated. Hnrnpdl exhibits a heat-included exon that leads to a PTC, Hnrnph3 a cold-skipped exon that leads to a frameshift, and in Cirbp, heat leads to inclusion of an alternative transcript end, which leads to formation of a PTC. (B) For each gene from A, the triplicate normalized read counts in DMSO (left y-axis, green) and the percentage of the poison isoform in CHX (right y-axis, orange) are plotted at the two temperatures. Line represents mean PSI.  (C) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature-dependent AS-decay skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform between the CHX samples. Top shows the GE change between the DMSO samples and bottom between the CHX samples. R² and P (deviation from zero slope) are indicated. N=24.  (E) Splicing reporters, containing only an intron (GFP ctrl, #1) or additionally temperature-dependent exons with ~1kB surrounding introns. Representative radioactive RT-PCRs (bottom) confirm cold-induced inclusion of Ythdf3 exon 7 (#2) and human Synerg exon 21 (#3) after 24 hours at the indicated temperatures. Representative images are derived from three independent gels. See SourceDataForFigure2E for uncropped versions of these gels.  (F-G) GFP expression was quantified using FACS and is shown relative to GFP ctrl. In (F) transfected cells were incubated at the indicated temperatures for 24 hours (n=3, mean +/- SD). In (G) cells were incubated directly after transfection for 12 hours at 39°C (blue) or 33°C (red) and then shifted for 2, 4, 8 and 12 hours (n=3, mean +/- SD).


Figure 3 ( A ) SR protein specific temperature - dependent AS - decay . Sashimi plots are shown for heat - induced poison exon inclusion for Srsf2 ( left ) and cold - induced poison isoform formation for Srsf10 ( right ) . Quantification of GE and AS are presented in the center ( as in Figure 2B ) , lines represent mean . ( B ) Poison exon inclusion for Srsf2 ( left ) and Srsf10 ( right ) in a 24h temperature - rhythm in human cells . Hek293 cells were pre - entrained with square - wave temperature cycles ( 12h 34 ° C / 12h 38 ° C ) for 48h . For the last 24h , cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT - PCR ( n = 3 , mean ± SD ) . White area : 34 ° C ; Red area : 38 ° C . In Hek293 cells , inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target . ( C ) AS - decay of human targets at 34 ° C ( blue ) and 38 ° C ( red ) was investigated after a square - wave temperature regime ( see B ) . RNAs were harvested after 56 and 68 hours , respectively ( n = 3 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA and is indicated by asterisks : adjusted p values : not significant ( ns ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . Individual data points are shown as circles . ( E ) Quantification of Srsf2 AS - decay in hamster , rabbit and chicken cells ( n = 2 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . Individual data points are shown as circles .

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

Figure 3 (A) SR protein specific temperature-dependent AS-decay. Sashimi plots are shown for heat-induced poison exon inclusion for Srsf2 (left) and cold-induced poison isoform formation for Srsf10 (right). Quantification of GE and AS are presented in the center (as in Figure 2B), lines represent mean.  (B) Poison exon inclusion for Srsf2 (left) and Srsf10 (right) in a 24h temperature-rhythm in human cells. Hek293 cells were pre-entrained with square-wave temperature cycles (12h 34°C/ 12h 38°C) for 48h. For the last 24h, cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT-PCR (n=3, mean ± SD). White area: 34°C; Red area: 38°C. In Hek293 cells, inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target.  (C) AS-decay of human targets at 34°C (blue) and 38°C (red) was investigated after a square-wave temperature regime (see B). RNAs were harvested after 56 and 68 hours, respectively (n=3, mean ± SD). Statistical significance was determined by 2-way ANOVA and is indicated by asterisks: adjusted p values: not significant (ns), **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.  (E) Quantification of Srsf2 AS-decay in hamster, rabbit and chicken cells (n=2, mean ± SD). Statistical significance was determined by 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.


Figure 4 Rhythmic Srsf2 productive mRNA levels ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h , Rhythmic Srsf10 productive mRNA levels ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated ( ∆ E3 A and B ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * * p < 0 . 01 , * * * * p < 0 . 0001 . Comparing temperature - induced expression of the core clock components Bmal1 in WT and Srsf10 ∆ E3 cells ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± SEM . Comparing temperature - induced expression of the core clock components Per1 in WT and Srsf10 ∆ E3 cells ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± SEM .

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

Figure 4 Rhythmic Srsf2 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h,  Rhythmic Srsf10 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated (∆E3 A and B). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: **p<0.01, ****p<0.0001.  Comparing temperature-induced expression of the core clock components Bmal1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.  Comparing temperature-induced expression of the core clock components Per1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.


Figure 5 ( A ) Normalized GE values of plant SR proteins in a 48 - hour time course . In the first day , plants were kept at 20 ° C ( red ) and shifted to 4 ° C ( blue ) for the second day . Plants were under a 12 - hour dark / light regimen ( gray / yellow bars ) . Warm - and cold - expressed genes are indicated . ( C ) Sashimi plots as shown in Figure 2A ( without conservation score ) for decay - inducing AS events upon cold ( At - SCL33 , left ) or warm temperature ( At - SR34 , right ) . Data shown represent , from top to bottom , warm - dark , warm - light , cold - dark and cold - light conditions as indicated on the right of the plots . ( D ) For validation , A . thaliana were incubated for 3 days either at 20 ° C or 4 ° C and RNA was investigated for gene expression ( green , left y - axis ) and poison isoform formation ( orange , right y - axis ) . Gene expression of At - SCL33 ( left ) or At - SR34 ( right ) is shown relative to Ipp2 . Lines represent mean , individual data points are shown . ( E ) Correlation of poison isoform inclusion and GE levels for temperature - dependent poison skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform . Decay targets were identified by using data from CHX - treated plants . R² and P ( deviation from zero slope ) are indicated . N = 80

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

Figure 5 (A) Normalized GE values of plant SR proteins in a 48-hour time course. In the first day, plants were kept at 20°C (red) and shifted to 4°C (blue) for the second day. Plants were under a 12-hour dark/light regimen (gray/yellow bars). Warm- and cold-expressed genes are indicated.  (C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots. (D) For validation, A. thaliana were incubated for 3 days either at 20°C or 4°C and RNA was investigated for gene expression (green, left y-axis) and poison isoform formation (orange, right y-axis). Gene expression of At-SCL33 (left) or At-SR34 (right) is shown relative to Ipp2. Lines represent mean, individual data points are shown.  (E) Correlation of poison isoform inclusion and GE levels for temperature-dependent poison skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform. Decay targets were identified by using data from CHX-treated plants. R² and P(deviation from zero slope) are indicated. N=80


Figure 6 ( B ) Correlation of rhythmic Srsf10 expression ( orange , left y - axis ) and exon 3 inclusion ( black , right y - axis ) in mouse liver samples from the indicated ZTs ( n = 4 , mean + / - SEM ) . Quantifications were obtained by RNA - seq analysis of datasets from ( Atger et al . , 2015 ) . ( C ) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts ( NX ) from different ZTs . The different SRSF10 variants are highlighted on the right . SRSF10 - fl and SRSF10 - 2 are the result of differential last exon usage . hnRNP L was used as a loading control . The asterisks could represent hyperphosphorylated SRSF10 - fl through higher CLK activity during the day . ( D ) Quantification of SRSF10 - fl + SRSF10 - 2 relative to ZT0 and hnRNP L ( mean of at least 3 mice ± SEM ) . Student ' s unpaired t test - derived p values * * * p < 0 . 001 .

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

Figure 6 (B) Correlation of rhythmic Srsf10 expression (orange, left y-axis) and exon 3 inclusion (black, right y-axis) in mouse liver samples from the indicated ZTs (n=4, mean +/- SEM). Quantifications were obtained by RNA-seq analysis of datasets from (Atger et al., 2015).  (C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day. (D) Quantification of SRSF10-fl + SRSF10-2 relative to ZT0 and hnRNP L (mean of at least 3 mice ± SEM). Student's unpaired t test-derived p values ***p<0.001.


Figure 1 ( A and B ) Root length ( averages ± SD , n ≥ 15 ) ( A ) and cell death staining of cotyledons and primary root tips ( B ) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days , 2 days after germination . BAK1 ( D416N ) is a kinase - dead variant . ( C ) Overview of 3 - week - old plants . ( D ) Pep2 induces PEPR1 - FLAG association with SERK1 - , SERK2 - , BKK1 - , and BAK1 - HA in N . benthamiana . Experiments were repeated at least three times , with the same conclusions . IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies , respectively . NT indicates non - transformed plants . ( * , p < 0 . 05 in two - tailed tests compared to the differences ( ± Pep2 ) from the corresponding values of WT plants . )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A and B) Root length (averages ± SD, n ≥ 15) (A) and cell death staining of cotyledons and primary root tips (B) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days, 2 days after germination. BAK1 (D416N) is a kinase-dead variant.(C) Overview of 3-week-old plants.(D) Pep2 induces PEPR1-FLAG association with SERK1-, SERK2-, BKK1-, and BAK1-HA in N. benthamiana. Experiments were repeated at least three times, with the same conclusions. IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies, respectively. NT indicates non-transformed plants. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants.)


Figure 2 ( A ) Immunoblot analysis for BIK1 - HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min . Arrowheads indicate phosphorylated BIK1 - HA . ( B ) Immunoblot analysis for Pep2 - triggered MAPK activation . Ponceau S - stained loading controls are shown ( bottom ) . ( C ) Root length ( averages ± SD , n ≥ 15 ) of Arabidopsis seedlings that were exposed to 100 nM Pep2 . ( * , p < 0 . 05 in two - tailed tests compared to the differences ( ± Pep2 ) from the corresponding values of bak1 - 3 plants . Two independent experiments were combined for statistical analysis . )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Immunoblot analysis for BIK1-HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min. Arrowheads indicate phosphorylated BIK1-HA.(B) Immunoblot analysis for Pep2-triggered MAPK activation. Ponceau S-stained loading controls are shown (bottom).(C) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings that were exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-3 plants. Two independent experiments were combined for statistical analysis.)


Figure 3 ( A ) Scatter plots for log2 fold changes of gene expression in WT plants versus bak1 - 3 plants treated with Pep2 for 2 and 10 h . The regression of these scatter plots is indicated by the red lines . ( B ) Hierarchical analysis of genes exhibiting a > 2 - fold change in expression level in bak1 - 3 plants after Pep2 application for 10 h in a whole - genome microarray analysis . Using Genevestigator v3 , these genes were separately cross - referenced to public databases to show their expression responses to salicylate ( SA ) , methyl - jasmonate ( JA ) , or ethylene ( ET ) . ( C and D ) qRT - PCR analysis of defense - related genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . Data are averages ( ± SD ) of three biological replicates . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding bak1 - 4 and bak1 - 3 values in C and D , respectively . Relative cycle threshold ( Ct ) values of two independent experiments were combined for statistical analysis . )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Scatter plots for log2 fold changes of gene expression in WT plants versus bak1-3 plants treated with Pep2 for 2 and 10 h. The regression of these scatter plots is indicated by the red lines.(B) Hierarchical analysis of genes exhibiting a >2-fold change in expression level in bak1-3 plants after Pep2 application for 10 h in a whole-genome microarray analysis. Using Genevestigator v3, these genes were separately cross-referenced to public databases to show their expression responses to salicylate (SA), methyl-jasmonate (JA), or ethylene (ET).(C and D) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding bak1-4 and bak1-3 values in C and D, respectively. Relative cycle threshold (Ct) values of two independent experiments were combined for statistical analysis.)


Figure 4 ( A ) qRT - PCR analysis of PROPEP genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis . ( B ) Anti - GFP ( upper ) and anti - PROPEP3 ( middle ) immunoblot analysis of PROPEP3 - Venus in seedlings submerged in liquid medium and treated with ( + ) or without ( - ) 0 . 5 M Pep2 for the indicated times . The positions of the molecular weight markers are shown on the left . Arrowheads and asterisks indicate PROPEP3 - Venus and cross - reactive bands , respectively . ( C , D and E ) Immunoblot analysis of PEPR1 - FLAG in 10 - day - old seedlings following Pep1 or flg22 application . ( C and D ) Plants were exposed to 1 M Pep1 or flg22 for the indicated times . ( E ) Plants were pre - exposed to MG132 ( 50 µM ) , Wortmannin ( Wm ) ( 30 µM ) and Tyrphostin A23 ( TyrA23 ) ( 100 µM ) for 1 h before being exposed to Pep1 for 10 h . The numbers below the immunoblots represent relative intensities of the PROPEP - Venus ( B ) and PEPR1 - FLAG ( C - E ) bands normalized to the backgrounds in the Ponceau S - stained loading controls ( bottom ) , with the band / background values in WT plant extracts 24 h ( upper ) and 0 h ( middle ) after Pep2 application , respectively , set as 1 . 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) qRT-PCR analysis of PROPEP genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.(B) Anti-GFP (upper) and anti-PROPEP3 (middle) immunoblot analysis of PROPEP3-Venus in seedlings submerged in liquid medium and treated with (+) or without (-) 0.5 M Pep2 for the indicated times. The positions of the molecular weight markers are shown on the left. Arrowheads and asterisks indicate PROPEP3-Venus and cross-reactive bands, respectively.(C, D and E) Immunoblot analysis of PEPR1-FLAG in 10-day-old seedlings following Pep1 or flg22 application. (C and D) Plants were exposed to 1 M Pep1 or flg22 for the indicated times. (E) Plants were pre-exposed to MG132 (50 µM), Wortmannin (Wm) (30 µM) and Tyrphostin A23 (TyrA23) (100 µM) for 1 h before being exposed to Pep1 for 10 h. The numbers below the immunoblots represent relative intensities of the PROPEP-Venus (B) and PEPR1-FLAG (C-E) bands normalized to the backgrounds in the Ponceau S-stained loading controls (bottom), with the band/background values in WT plant extracts 24 h (upper) and 0 h (middle) after Pep2 application, respectively, set as 1.0.


Figure 5 ( A ) qRT - PCR analysis of defense - related genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis . ) ( B ) Root length ( averages ± SD , n ≥ 15 ) of Arabidopsis seedlings exposed to 100 nM Pep2 . ( * , p < 0 . 05 in two - tailed tests compared to the differences ( ± Pep2 ) from the corresponding values of WT plants . Two independent experiments were combined for statistical analysis . ) ( C ) Fresh weight ( averages ± SD , n ≥ 25 ) of 2 - week - old plants grown on soil . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding values of WT plants . ■ , p < 0 . 05 in two - tailed tests . Two independent experiments were combined for statistical analysis . ) ( D ) Immunoblot analysis for BAK1 in 10 - day - old seedlings with or without 0 . 5 M Pep1 for 24 h . The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S - stained loading controls , with the value in non - elicited WT plants set as 1 . 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.)(B) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)(C) Fresh weight (averages ± SD, n ≥ 25) of 2-week-old plants grown on soil. (*, p < 0.05 in two-tailed tests compared to the corresponding values of WT plants. ■, p < 0.05 in two-tailed tests. Two independent experiments were combined for statistical analysis.)(D) Immunoblot analysis for BAK1 in 10-day-old seedlings with or without 0.5 M Pep1 for 24 h. The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S-stained loading controls, with the value in non-elicited WT plants set as 1.0.


Figure 6 ( A ) qRT - PCR analysis in 4 - week - old plant leaves challenged with the indicated P . syringae pv . tomato ( Pst ) DC3000 strains for the indicated times . ( B ) Anti - GFP immunoblot analyses in seedlings challenged with Pst DC3000 ( DC3000 ) and Pst DC3000 ∆ hrcC ( ∆ hrcC ) for the indicated times . . ( C ) Anti - PROPEP3 immunoblot analysis of PROPEP3 - Venus ( upper ) in plant tissues ( expressed in plants ) and in the extracellular fraction ( released into medium ) , 24 h and 48 h after the inoculation with ( + ) or without ( - ) Pst DC3000 . Lower panels indicate Ponceau S - stained loading controls . The numbers below the upper panel represent relative intensities of the PROPEP3 - Venus band normalized to the backgrounds in the Ponceau S - stained loading controls . NT denotes non - transgenic WT plants . ( D ) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 ( AvrRpm1 ) for 48 h . ( E ) Anti - PROPEP3 immunoblot analyses in the seedlings exposed to the indicated Pst DC3000 strains or 1 M Pep1 for 48 h . ( F ) Anti - GFP immunoblot analysis for the extracellular fraction of the seedlings exposed to Pst DC3000 AvrRpm1 for 48 h . ( B , C , E and F ) Arrowheads and arrows indicate an apparently full - length and processed form of PROPEP3 - Venus , respectively . Asterisks indicate cross - reactive bands . The positions of the molecular weight markers are shown on the left of the immunoblots .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) qRT-PCR analysis in 4-week-old plant leaves challenged with the indicated P. syringae pv. tomato (Pst) DC3000 strains for the indicated times.(B) Anti-GFP immunoblot analyses in seedlings challenged with Pst DC3000 (DC3000) and Pst DC3000 ∆hrcC (∆hrcC) for the indicated times.. (C) Anti-PROPEP3 immunoblot analysis of PROPEP3-Venus (upper) in plant tissues (expressed in plants) and in the extracellular fraction (released into medium), 24 h and 48 h after the inoculation with (+) or without (-) Pst DC3000. Lower panels indicate Ponceau S-stained loading controls. The numbers below the upper panel represent relative intensities of the PROPEP3-Venus band normalized to the backgrounds in the Ponceau S-stained loading controls. NT denotes non-transgenic WT plants.(D) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 (AvrRpm1) for 48 h.(E) Anti-PROPEP3 immunoblot analyses in the seedlings exposed to the indicated Pst DC3000 strains or 1 M Pep1 for 48 h.(F) Anti-GFP immunoblot analysis for the extracellular fraction of the seedlings exposed to Pst DC3000 AvrRpm1 for 48 h. (B, C, E and F) Arrowheads and arrows indicate an apparently full-length and processed form of PROPEP3-Venus, respectively. Asterisks indicate cross-reactive bands. The positions of the molecular weight markers are shown on the left of the immunoblots.


Figure 7 ( A ) Growth of H . arabidopsidis ( Hpa ) Noco2 in 2 - week - old plants . Spores were counted at 6 days post inoculation ( dpi ) . Results are averages ± SE of three biological replicates . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding values of Col plants . ■ , p < 0 . 05 in two - tailed tests between the indicated values . Three independent experiments were combined for statistical analysis . ) ( B ) Growth of syringe - infiltrated Pst DC3000 in rosette leaves of 4 - week - old plants pretreated with water ( Mock ) or 1 M flg22 for 24 h . Bacterial titers were determined at 3 dpi . Results are averages ± SE of three independent replicates . ( * , p < 0 . 05 , * * , p < 0 . 01 in two - tailed tests compared to the corresponding values of WT plants . ▲ , p < 0 . 01 in two - tailed tests compared to the differences ( ± flg22 ) from the corresponding values of WT plants . Three independent experiments were combined for statistical analysis . ) ( C ) Total SA levels were determined in the rosette leaves of 4 - week - old plants with or without 1 µM flg22 application for 24 h . Data are averages ( ± SD ) of material from three independent plants . The experiments were repeated twice , with similar results . ( ▲ , p < 0 . 01 in two - tailed tests compared to the differences ( ± flg22 ) from the corresponding values of WT plants . Two independent experiments were combined for statistical analysis . ) ( D ) qRT - PCR analysis of the PR1 gene in 4 - week - old plants exposed to 1 µM flg22 . Data are averages ( ± SD ) of three biological replicates . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Relative Ct values of four independent experiments were combined for statistical analysis . ) ( E ) Growth of syringe - infiltrated Pst DC3000 in rosette leaves of 4 - week - old plants pretreated with water ( Mock ) or 1 M flg22 for 24 h . Bacterial titers were determined at 3 dpi . Results are averages ± SE of three independent replicates . ( * * , p < 0 . 01 , * * * , p < 1 . 0e - 10 in two - tailed tests compared to the corresponding values of WT plants . ▲ , p < 0 . 01 in two - tailed tests compared to the differences ( ± flg22 ) from the corresponding values of WT plants . Three independent experiments were combined for statistical analysis . )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Growth of H. arabidopsidis (Hpa) Noco2 in 2-week-old plants. Spores were counted at 6 days post inoculation (dpi). Results are averages ± SE of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding values of Col plants. ■, p < 0.05 in two-tailed tests between the indicated values. Three independent experiments were combined for statistical analysis.)(B) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (*, p < 0.05, **, p < 0.01 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)(C) Total SA levels were determined in the rosette leaves of 4-week-old plants with or without 1 µM flg22 application for 24 h. Data are averages (±SD) of material from three independent plants. The experiments were repeated twice, with similar results. (▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)(D) qRT-PCR analysis of the PR1 gene in 4-week-old plants exposed to 1 µM flg22. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of four independent experiments were combined for statistical analysis.)(E) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (**, p < 0.01, ***, p < 1.0e-10 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)


Figure 8 ( A ) Lesion sizes ( left ) were determined 5 days after C . higginsianum ( Ch ) inoculation . Invasion rates ( right ) of Ch on Arabidopsis leaves were determined 3 days after spore inoculation . An average of 3 replicates is shown ( ± SD ) ( left ) and ( ± SE ) ( right ) . ( ■ , p < 0 . 05 in two - tailed tests between the indicated samples . * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Two independent experiments were combined for statistical analysis . ) B ) Immunoblot ( left ) and qRT - PCR ( right ) analyses following Ch challenge for the indicated times ( dpi ) . The numbers below the immunoblots represent relative intensities of the indicated bands normalized to the backgrounds , with the values at 0 dpi set as 1 . 0 . Experiments were performed three times with the same conclusions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Lesion sizes (left) were determined 5 days after C. higginsianum (Ch) inoculation. Invasion rates (right) of Ch on Arabidopsis leaves were determined 3 days after spore inoculation. An average of 3 replicates is shown (±SD) (left) and (±SE) (right). (■, p < 0.05 in two-tailed tests between the indicated samples. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Two independent experiments were combined for statistical analysis.)B) Immunoblot (left) and qRT-PCR (right) analyses following Ch challenge for the indicated times (dpi). The numbers below the immunoblots represent relative intensities of the indicated bands normalized to the backgrounds, with the values at 0 dpi set as 1.0. Experiments were performed three times with the same conclusions.


Figure 1E . Microscale thermophoresis binding experiment for PlexA1 - 4 - mVenus and Sema1becto ( red ) or Sema1becto - mutA ( blue ) or Sema1becto - mutB ( green , ) or Sema1becto - mutA + B ( orange ) . The Sema1becto wild - type binding results are as reported previously ( Rozbesky et al . , 2019 ) . All data were collected at the same time , error bars represent s . d . of three technical replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.


Figure 2A . FRET - FLIM in live COS - 7 cells shows cell surface PlexA - Sema1b cis interaction . Sema1b mutants reveal that both head - to - head and side - on orientations of the PlexA - Sema1b complex are involved in the cis interaction . The cis interaction between Sema1a and PlexA is not observed . The boxcharts represent the average lifetime . Box limits indicate the 25th and 75th percentiles , centred lines show the median , squares represent sample means , whiskers extend 1 . 5 - fold the interquartile range from the 25th and 75th percentiles , p - value was calculated by one - way analysis of variance ( ANOVA ) . * P < 0 . 05 , * * * P < 0 . 001B . Representative negative stain class averages of PlexAecto correlate well with the ring - like conformation of mouse PlxnA1 . The ring - like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue ( N terminus ) to red ( C terminus ) . Scale bar , 10 nm . E . In vitro FRET - FLIM measurement of the PlexA - FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation . The boxcharts represent the average lifetime . Box limits indicate the 25th and 75th percentiles , centred lines show the median , squares represent sample means , whiskers extend 1 . 5 - fold the interquartile range from the 25th and 75th percentiles , p - value was calculated by one - way analysis of variance ( ANOVA ) . * * * P < 0 . 001 . F . Relative change of COS - 7 cells surface area upon treatment with Sema1a - Fc . Cells expressing PlexAFL - mClover or PlexAFL - mCover and Sema1b - mRuby2 wild - type or mutants were treated with purified Sema1a - Fc at a final concentration of 5 . 8 µM . Images were acquired every minute for 30 minutes . Cell surface area was calculated using ImageJ before and after stimulation . Data are presented as means ± sem . * P < 0 . 05 , * * * P < 0 . 001 , p - value calculated by one - way analysis of variance ( ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. FRET-FLIM in live COS-7 cells shows cell surface PlexA-Sema1b cis interaction. Sema1b mutants reveal that both head-to-head and side-on orientations of the PlexA-Sema1b complex are involved in the cis interaction. The cis interaction between Sema1a and PlexA is not observed. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). *P<0.05, ***P<0.001B. Representative negative stain class averages of PlexAecto correlate well with the ring-like conformation of mouse PlxnA1. The ring-like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue (N terminus) to red (C terminus). Scale bar, 10 nm.E. In vitro FRET-FLIM measurement of the PlexA-FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). ***P<0.001.F. Relative change of COS-7 cells surface area upon treatment with Sema1a-Fc. Cells expressing PlexAFL-mClover or PlexAFL-mCover and Sema1b-mRuby2 wild-type or mutants were treated with purified Sema1a-Fc at a final concentration of 5.8 µM. Images were acquired every minute for 30 minutes. Cell surface area was calculated using ImageJ before and after stimulation. Data are presented as means ± sem. *P<0.05, ***P<0.001, p-value calculated by one-way analysis of variance (ANOVA).