G Relative expression levels of IFNB mRNA and interferon - stimulated genes , including ISG54 and ISG56 in WT , BECN1 , ATG5 and SQSTM1 KO cells with or without USP19 .

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G Relative expression levels of IFNB mRNA and interferon-stimulated genes, including ISG54 and ISG56 in WT, BECN1, ATG5 and SQSTM1 KO cells with or without USP19.


H , I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE ( H ) or IFN - β ( I ) promoter - driven luciferase reporter after infection with SeV for indicated time points .

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H, I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (H) or IFN-β (I) promoter-driven luciferase reporter after infection with SeV for indicated time points.


A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE ( left ) or IFN - β ( right ) promoter - driven luciferase reporter after infection with SeV for 12 h .

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A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter after infection with SeV for 12 h.


B WT and BECN1 KO 293T cells were infected with SeV for indicated time points , then protein extracts were analyzed by immunoblot using the indicated antibodies .

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B WT and BECN1 KO 293T cells were infected with SeV for indicated time points, then protein extracts were analyzed by immunoblot using the indicated antibodies.


C Protein extracts of THP - 1 cells infected with SeV for various time points ( upper lanes ) were subjected to immunoprecipitation with anti - Beclin - 1 and immunoblot analysis with antibodies shown on the left .

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C Protein extracts of THP-1 cells infected with SeV for various time points (upper lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with antibodies shown on the left.


D Co - immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV .

D Co-immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV.


E 293T cells were transfected with vectors encoding HA - Beclin - 1 and Flag - MAVS or Flag - MAVSΔCARD , followed by IP with anti - Flag beads and immunoblot analysis with anti - HA .

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

E 293T cells were transfected with vectors encoding HA-Beclin-1 and Flag-MAVS or Flag-MAVSΔCARD, followed by IP with anti-Flag beads and immunoblot analysis with anti-HA.


F Co - immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA - MAVS plasmid along with vector encoding Flag - RIG - I ( N ) in the absence or presence of USP19 .

F Co-immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA-MAVS plasmid along with vector encoding Flag-RIG-I (N) in the absence or presence of USP19.


G Protein extracts of scramble or USP19 - specific siRNA treated 293T cells transfected with HA - MAVS plasmid along with vector encoding for Flag - RIG - I ( N ) in the absence or presence of Beclin - 1 were subjected to immunoprecipitation and immunoblot analysis .

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

G Protein extracts of scramble or USP19-specific siRNA treated 293T cells transfected with HA-MAVS plasmid along with vector encoding for Flag-RIG-I (N) in the absence or presence of Beclin-1 were subjected to immunoprecipitation and immunoblot analysis.


H Luciferase activity in 293T cells transfected with scramble or USP19 - specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 .

H Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin-1.


I Luciferase activity in 293T cells transfected with scramble or USP19 - specific siRNA and IFN - β luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 .

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

I Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and IFN-β luciferase reporter after SeV infection for indicated time points with or without Beclin-1.


( A ) Expression patterns of Hoxa5 and Hoxc8 in MNs ( demarcated by white dashed lines ) along the rostrocaudal axis of the spinal cord ( cervical C4 to thoracic T1 ) from mouse embryos from E9 . 5 to E12 . 5 . Pink scale bars : 12 . 5 μm for E9 . 5 ; 12 . 5 μm for E10 . 5 ; 25 μm for E11 . 5 ; and 50 μm for E12 . 5 .

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(A) Expression patterns of Hoxa5 and Hoxc8 in MNs (demarcated by white dashed lines) along the rostrocaudal axis of the spinal cord (cervical C4 to thoracic T1) from mouse embryos from E9.5 to E12.5. Pink scale bars: 12.5 μm for E9.5; 12.5 μm for E10.5; 25 μm for E11.5; and 50 μm for E12.5.


Expression patterns of Hoxa5 and Hoxc8 mRNAs , as revealed by in situ hybridization , aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs ( demarcated with pink / white dashed lines ) along the cervical spinal cord ( cervical C5 to C7 ) in mouse embryos at E12 . 5 . Pink scale bar , 50 μm . White and pink dash lines demarcate the postmitotic MN region .

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

Expression patterns of Hoxa5 and Hoxc8 mRNAs, as revealed by in situ hybridization, aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs (demarcated with pink/white dashed lines) along the cervical spinal cord (cervical C5 to C7) in mouse embryos at E12.5. Pink scale bar, 50 μm. White and pink dash lines demarcate the postmitotic MN region.


Dot - plots showing expression of genes ( rows ) that distinguish MN subtypes ( columns ) . The height of each colored shape is proportional to the percentage of cells expressing the marker ( ≥ 1 UMI ) , and its width reflects the average transcript count within expressing cells .

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

Dot-plots showing expression of genes (rows) that distinguish MN subtypes (columns). The height of each colored shape is proportional to the percentage of cells expressing the marker (≥1 UMI), and its width reflects the average transcript count within expressing cells.


Immunostaining ( C ) of EBs 96 h after ESC differentiation , and quantification ( D ) of Hoxa5on in MNs ( Hb9on ) .

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Immunostaining (C) of EBs 96 h after ESC differentiation, and quantification (D) of Hoxa5on in MNs (Hb9on).


Hoxa5 expression upon miR - 27 knockdown , with exposure to RAhi for 72 h , or with exposure to RAhi for 48 h , and then switching to RAlow plus RAR inhibitor ( 1 µM ) for a further 24 h , or with exposure to RAmed for 72 h .

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

Hoxa5 expression upon miR-27 knockdown, with exposure to RAhi for 72 h, or with exposure to RAhi for 48 h, and then switching to RAlow plus RAR inhibitor (1 µM) for a further 24 h, or with exposure to RAmed for 72 h.


( C ) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6 / 7 segments of miR - 23 ~ 27 ~ 24 double KO ( DKO ) mice . miR - 23a ~ 27a ~ 24 - 2 + / - ; miR - 23b ~ 27b ~ 24 - 1 - / - mice were used as a control ( ctrl ) . ( E ) Conversely , Hoxc8 is shifted into the MNs of cervical C5 segments of both miR - 196a2 / b DKO and miR - 196a1 / a2 / b triple knockout ( TKO ) embryos . Age - matched wild type embryos were used as controls . Scale bar represents 50 μm . White dash lines demarcate the postmitotoic MN domain ( D , F ) Quantifications of Hoxa5on , Hoxc8on , and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos ( mean ± SD , N = 3 embryos , * p < 0 . 01 , Student ' s t - tests ) .

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(C) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6/7 segments of miR-23~27~24 double KO (DKO) mice. miR-23a~27a~24-2+/-;miR-23b~27b~24-1-/- mice were used as a control (ctrl). (E) Conversely, Hoxc8 is shifted into the MNs of cervical C5 segments of both miR-196a2/b DKO and miR-196a1/a2/b triple knockout (TKO) embryos. Age-matched wild type embryos were used as controls. Scale bar represents 50 μm. White dash lines demarcate the postmitotoic MN domain (D, F) Quantifications of Hoxa5on, Hoxc8on, and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos (mean ± SD, N=3 embryos, *p < 0.01, Student's t-tests ).


G . Relative expression levels of shade - regulated lncRNAs by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 20 µE , B = 20 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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G. Relative expression levels of shade-regulated lncRNAs by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 20 µE, B = 20 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


Relative expression levels of PUAR under WL , SH , and dark ( DK ) conditions determined by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . The seedlings were grown under DK for 4 d or transferred to WL for 6 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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Relative expression levels of PUAR under WL, SH, and dark (DK) conditions determined by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. The seedlings were grown under DK for 4 d or transferred to WL for 6 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by t-test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


Relative expression levels of PUAR in Col - 0 , phyB - 9 , phyA - 211 , and pif7 - 1 by RT - qPCR under Red ( R = 100 µE ) , Far - red ( FR = 10 µE ) , or SH conditions . For R and FR conditions , the seedlings were grown under R or FR for 4 d . For SH conditions , the seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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Relative expression levels of PUAR in Col-0, phyB-9, phyA-211, and pif7-1 by RT-qPCR under Red (R = 100 µE), Far-red (FR = 10 µE), or SH conditions. For R and FR conditions, the seedlings were grown under R or FR for 4 d. For SH conditions, the seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by t-test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


Hypocotyl phenotypes of Col - 0 , PUARox , PUAR - S1ox , The seedlings were grown under white light for 4 d , and then maintained under white light or transferred to shade for 3 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . The scale bar represents 2 mm . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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Hypocotyl phenotypes of Col-0, PUARox, PUAR-S1ox, The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).


Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions . The scale bar represents 2 mm . P - values indicate statistically significant differences ( * P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions. The scale bar represents 2 mm. P-values indicate statistically significant differences (*P-value < 0.05) by t-test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).


Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions . The scale bar represents 2 mm . P - values indicate statistically significant differences ( * P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) . J . Hypocotyl phenotypes of Col - 0 , PHYAox , and amiR - puar . The seedlings were grown under white light for 3 d , and then maintained under white light or transferred to shade for 4 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . The scale bar represents 2 mm . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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

Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions. The scale bar represents 2 mm. P-values indicate statistically significant differences (*P-value < 0.05) by t-test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates). J. Hypocotyl phenotypes of Col-0, PHYAox, and amiR-puar. The seedlings were grown under white light for 3 d, and then maintained under white light or transferred to shade for 4 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).


Relative expression levels of PHYA in Col - 0 , pif7 - 1 , PUARox , PUAR - S1ox , by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . The left Y - axis is relative expression level of PUAR , and the right Y - axis is the ratio of relative expression level under shade conditions to that under white light conditions . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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Relative expression levels of PHYA in Col-0, pif7-1, PUARox, PUAR-S1ox, by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. The left Y-axis is relative expression level of PUAR, and the right Y-axis is the ratio of relative expression level under shade conditions to that under white light conditions. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


PHYA protein levels in Col - 0 , pif7 - 1 , PUARox , PUAR - S1ox , by western - blot . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Tubulin indicates the loading control .

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PHYA protein levels in Col-0, pif7-1, PUARox, PUAR-S1ox, by western-blot. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Tubulin indicates the loading control.


Relative expression levels of PHYA in Col - 0 TATA - mut , and amiR - puar by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . The left Y - axis is relative expression level of PUAR , and the right Y - axis is the ratio of relative expression level under shade conditions to that under white light conditions . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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

Relative expression levels of PHYA in Col-0 TATA-mut, and amiR-puar by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. The left Y-axis is relative expression level of PUAR, and the right Y-axis is the ratio of relative expression level under shade conditions to that under white light conditions. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


PHYA protein levels in Col - 0 , TATA - mut , and amiR - puar by western - blot . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Tubulin indicates the loading control .

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

PHYA protein levels in Col-0, TATA-mut, and amiR-puar by western-blot. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Tubulin indicates the loading control.


G - H . Hypocotyl phenotypes of Col - 0 , phyA - 211 , PUARox , PUAR - S1ox , PUARox / phyA - 211 , GFPox , PHYAox , PUAR - S1ox , and PHYAox * PUAR - S1ox . The seedlings were grown under white light for 4 d , and then maintained under white light or transferred to shade for 3 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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

G-H. Hypocotyl phenotypes of Col-0, phyA-211, PUARox, PUAR-S1ox, PUARox/phyA-211, GFPox, PHYAox, PUAR-S1ox, and PHYAox*PUAR-S1ox. The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).


Detection of PUAR in the nuclear and non - nuclear fractions by RT - qPCR . U6 and tRNA were used as RNA markers for the nuclear and non - nuclear fractions , respectively . The seedlings were grown under white light for 6 d , and then maintained under white light , or transferred to shade for 4 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 4 , n refers to technical replicates ) .

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

Detection of PUAR in the nuclear and non-nuclear fractions by RT-qPCR. U6 and tRNA were used as RNA markers for the nuclear and non-nuclear fractions, respectively. The seedlings were grown under white light for 6 d, and then maintained under white light, or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 4, n refers to technical replicates).


Remaining levels of PHYA and GAPDH at different time points post 1 mM cordycepin in Col - 0 and PUAR - S1ox lines . 6 d - old seedlings were shade - treated for 4 h prior to cordycepin treatment . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Data are presented as means ± SD ( n = 3 , n refers to biological replicates ) .

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

Remaining levels of PHYA and GAPDH at different time points post 1 mM cordycepin in Col-0 and PUAR-S1ox lines. 6 d-old seedlings were shade-treated for 4 h prior to cordycepin treatment. SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Data are presented as means ± SD (n = 3, n refers to biological replicates).


PIF7 specifically binds to PUAR in vitro . Purified TF - His - PIF7 from E . coli was incubated with the biotinylated PUAR at 4 ° C for 1 h , and then non - biotinylated PUAR was added according to the dose as shown in the figure at 4 ° C for 1 h . These reaction systems were further incubated with streptavidin - coupled agarose beads in the reaction mix to isolate RNA complex at 4 ° C for 2 h .

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

PIF7 specifically binds to PUAR in vitro. Purified TF-His-PIF7 from E. coli was incubated with the biotinylated PUAR at 4 °C for 1 h, and then non-biotinylated PUAR was added according to the dose as shown in the figure at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate RNA complex at 4 °C for 2 h.


Interaction between PUAR and PIF7 was determined using an in vivo pull - down assay . RNA - protein extracts were from plants that overexpressed PIF7 - Flash * Col - 0 and PIF7 - Flash * PUAR - S1ox , respectively . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Streptavidin - coupled agarose beads were added in the reaction mix to isolate RNA complex for 2 h .

Interaction between PUAR and PIF7 was determined using an in vivo pull-down assay. RNA-protein extracts were from plants that overexpressed PIF7-Flash*Col-0 and PIF7-Flash*PUAR-S1ox, respectively. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Streptavidin-coupled agarose beads were added in the reaction mix to isolate RNA complex for 2 h.


Relative expression levels of PHYA in Col - 0 , pif7 - 1 , and PIF7 - Flash by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) . Relative expression levels of PHYA in Col - 0 , phyB - 9 , and pif7 - 1 * phyB - 9 by RT - qPCR . The seedlings were grown under white light for 6 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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

Relative expression levels of PHYA in Col-0, pif7-1, and PIF7-Flash by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).   Relative expression levels of PHYA in Col-0, phyB-9, and pif7-1*phyB-9 by RT-qPCR. The seedlings were grown under white light for 6 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


PIF7 specifically binds to 5 ′ UTR of the PHYA in vitro . Purified His - PIF7 from E . coli was incubated with the biotinylated 5 ′ UTR of the PHYA fragment at 4 ° C for 1 h , and then non - biotinylated 5 ′ UTR of the PHYA fragment was added according to the dose , as shown in the figure , at 4 ° C for 1 h . These reaction systems were further incubated with streptavidin - coupled agarose beads in the reaction mix to isolate DNA complex for at 4 ° C for 1 h .

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

PIF7 specifically binds to 5′ UTR of the PHYA in vitro. Purified His-PIF7 from E. coli was incubated with the biotinylated 5′ UTR of the PHYA fragment at 4 °C for 1 h, and then non-biotinylated 5′ UTR of the PHYA fragment was added according to the dose, as shown in the figure, at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate DNA complex for at 4 °C for 1 h.


Hypocotyl phenotypes of Col - 0 , pif7 - 1 , phyA - 211 , and pif7 - 1 * phyA - 211 under WL and SH . The seedlings were grown under white light for 4 d , and then maintained under white light or transferred to shade for 3 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . The scale bar represents 2 mm . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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

Hypocotyl phenotypes of Col-0, pif7-1, phyA-211, and pif7-1*phyA-211 under WL and SH. The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).


Hypocotyl phenotypes of Col - 0 , pif7 - 1 , PUARox , and PUARox / pif7 - 1 . The seedlings were grown under white light for 4 d , and then maintained under white light or transferred to shade for 3 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . The scale bar represents 2 mm . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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

Hypocotyl phenotypes of Col-0, pif7-1, PUARox, and PUARox/pif7-1. The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).


Relative expression levels of PHYA in Col - 0 , pif7 - 1 , PUARox , and PUARox / pif7 - 1 by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

Relative expression levels of PHYA in Col-0, pif7-1, PUARox, and PUARox/pif7-1 by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


PUAR specifically blocks the binding of PIF7 to the 5 ′ UTR of PHYA in vitro . Purified TF - His - PIF7 from E . coli was incubated with the biotinylated 5 ′ UTR of the PHYA fragment or fragment of the PIL1 promoter at 4 ° C for 1 h , and then PUAR or anti - PUAR was added according to the dose as shown in the figure , at 4 ° C for 1 h . These reaction systems were further incubated with streptavidin - coupled agarose beads in the reaction mix to isolate DNA complex for at 4 ° C for 1 h .

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

PUAR specifically blocks the binding of PIF7 to the 5′ UTR of PHYA in vitro. Purified TF-His-PIF7 from E. coli was incubated with the biotinylated 5′ UTR of the PHYA fragment or fragment of the PIL1 promoter at 4 °C for 1 h, and then PUAR or anti-PUAR was added according to the dose as shown in the figure, at 4 °C for 1 h. These reaction systems were further incubated with streptavidin-coupled agarose beads in the reaction mix to isolate DNA complex for at 4 °C for 1 h.


I - J . Relative expression levels of PHYA and PIL1 in Col - 0 , PIF7 - Flash , PUAR - S1ox , PIF7 - Flash * Col - 0 , and PIF7 - Flash * PUAR - S1ox by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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

I-J. Relative expression levels of PHYA and PIL1 in Col-0, PIF7-Flash, PUAR-S1ox, PIF7-Flash*Col-0, and PIF7-Flash*PUAR-S1ox by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


D . Immunoblot detection of the indicated proteins in whole cell extracts ( WCE ) , PrimPol immunoprecipitates ( IP ) , and non - precipitated flow - through fractions ( FT ) . IgG shows the presence of anti - PrimPol antibody in the IP fractions ( lanes 4 - 6 ) .

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

D. Immunoblot detection of the indicated proteins in whole cell extracts (WCE), PrimPol immunoprecipitates (IP), and non-precipitated flow-through fractions (FT). IgG shows the presence of anti-PrimPol antibody in the IP fractions (lanes 4-6).


B . Confocal microscopy images of PrimPol and γH2AX ( damage control ) immunofluorescence staining in control ( UVA ) or TMP - UVA - laser irradiated ( 10 µM TMP , 2 h followed by UVA laser irradiation ) WT and PRIMPOL KO cells . Nuclear DNA is counterstained with DAPI . UVA laser path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show the average percentage of PrimPol - positive cells and SD of three assays ( n ≥ 100 cells per TMP - treated conditions in each replicate ) . Circle dots in each column represent the values of individual replicates . Statistical analysis was conducted with one - way ANOVA followed by Bonferroni post - test . p - values of individual comparisons are indicated .

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

B. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-laser irradiated (10 µM TMP, 2 h followed by UVA laser irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


C . Confocal microscopy images of PrimPol and γH2AX ( damage control ) immunofluorescence staining in control ( UVA ) or TMP - UVA - treated cells following control or RPA2 downregulation . Nuclear DNA is counterstained with DAPI . UVA laser path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show the average PrimPol - positive cells and SD of three assays ( n ≥ 100 cells per TMP - treated conditions in each replicate ) . Circle dots in each column represent the values of individual replicates . Statistical analysis was conducted with one - way ANOVA followed by Bonferroni post - test . p - values of individual comparisons are indicated .

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

C. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated cells following control or RPA2 downregulation. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


D . Confocal microscopy images of PrimPol and γH2AX ( damage control ) immunofluorescence staining in control ( UVA ) or TMP - UVA - treated PRIMPOL KO cells stably expressing V5 - tagged WT or RPA binding domain mutant ( RBDm ) PrimPol versions . Nuclear DNA is counterstained with DAPI . UVA laser path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show the average percentage of PrimPol - positive cells and SD of two assays ( n ≥ 100 cells per condition in each replicate ) . Circle dots in each column represent the values of individual replicates . Statistical analysis was conducted with one - way ANOVA followed by Bonferroni post - test . p - values of individual comparisons are indicated .

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

D. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated PRIMPOL KO cells stably expressing V5-tagged WT or RPA binding domain mutant (RBDm) PrimPol versions. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of two assays (n ≥100 cells per condition in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


E . Confocal microscopy images of PrimPol and γH2AX ( damage control ) immunofluorescence staining in control ( UVA ) or TMP - UVA - treated WT and BLM KO cells upon downregulation of TOP3A , RMI1 and RMI2 ( siTR ) when indicated . Nuclear DNA is counterstained with DAPI . UVA laser path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show the average percentage of PrimPol - positive cells and SD of three assays ( n ≥ 250 cells per condition ) . Circle dots in each column represent the values of individual replicates . Statistical analysis was conducted with one - way ANOVA followed by Bonferroni post - test .

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

E. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated WT and BLM KO cells upon downregulation of TOP3A, RMI1 and RMI2 (siTR) when indicated. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥250 cells per condition). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test.


H . Immunoblots showing the levels of BLM , FANCD2 and PrimPol after BLM or FANCD2 downregulation in WT and PRIMPOL KO cells for experiments in ( G ) . Ponceau - S is shown as loading control .

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

H. Immunoblots showing the levels of BLM, FANCD2 and PrimPol after BLM or FANCD2 downregulation in WT and PRIMPOL KO cells for experiments in (G). Ponceau-S is shown as loading control.


C . Representative confocal microscopy images of EdU staining in WT and PRIMPOL KO cells , 8 h after UVA ( control ) or TMP - UVA treatment ( 2 µM TMP , 2 h followed by 5 s irradiation ) . Nuclear DNA is counterstained with DAPI . When indicated , V5 - PrimPol was reintroduced into KO cells . Scale bar , 25 µm . Dot plots indicate the distribution of nuclear EdU intensity , and the mean values are indicated by horizontal red lines . Data from the combination of three replicates ( n ≥ 200 cells per condition ) . Statistical analysis was conducted with Kruskal - Wallis test and Dunns post - test . p - values of individual comparisons are indicated .

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

C. Representative confocal microscopy images of EdU staining in WT and PRIMPOL KO cells, 8 h after UVA (control) or TMP-UVA treatment (2 µM TMP, 2 h followed by 5 s irradiation). Nuclear DNA is counterstained with DAPI. When indicated, V5-PrimPol was reintroduced into KO cells. Scale bar, 25 µm. Dot plots indicate the distribution of nuclear EdU intensity, and the mean values are indicated by horizontal red lines. Data from the combination of three replicates (n ≥200 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated.


A . Confocal microscopy images of FANCD2 immunofluorescence staining in control ( UVA ) or TMP - UVA - treated ( 2 µM TMP , 2 h followed by 5 s irradiation ) WT and PRIMPOL KO cells . Nuclear DNA is counterstained with DAPI . Scale bar , 25 µm .

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

A. Confocal microscopy images of FANCD2 immunofluorescence staining in control (UVA) or TMP-UVA-treated (2 µM TMP, 2 h followed by 5 s irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.


D . Histograms show the number of chromosomal aberrations per metaphase ( average and SEM ) in each condition . Data taken from three replicates are combined ( 50 metaphases per replicate ) . Statistical analysis was conducted with one - way ANOVA followed by Bonferroni post - test . p - values of individual comparisons are indicated .

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

D. Histograms show the number of chromosomal aberrations per metaphase (average and SEM) in each condition. Data taken from three replicates are combined (50 metaphases per replicate). Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


A . Kaplan - Meier survival curves of WT and PRIMPOL KO mice after the administration of a single dose of MMC ( 7 . 5 , 10 or 15 mg / kg ) . Dashed vertical lines represent median survival values for WT ( blue ) and KO ( orange ) mice . Statistical analysis was conducted with Log - rank ( Mantel - Cox ) test ; p - value for the highest dose of MMC ( 15 mg / kg ) is indicated .

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

A. Kaplan-Meier survival curves of WT and PRIMPOL KO mice after the administration of a single dose of MMC (7.5, 10 or 15 mg/kg). Dashed vertical lines represent median survival values for WT (blue) and KO (orange) mice. Statistical analysis was conducted with Log-rank (Mantel-Cox) test; p-value for the highest dose of MMC (15 mg/kg) is indicated.


B . Haematoxylin - eosin ( H & E ) and Ki - 67 immunohistochemistry ( IHC ) stainings of bone marrow sections derived from WT and PRIMPOL KO mice following the administration of 10 mg / kg MMC . Control mice ( PBS ) were sacrificed when MMC - injected mice reached the humane endpoint . Scale bar , 2 . 5 mm . In each image , a relevant area is shown at higher magnification ( 10x ) .

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

B. Haematoxylin-eosin (H&E) and Ki-67 immunohistochemistry (IHC) stainings of bone marrow sections derived from WT and PRIMPOL KO mice following the administration of 10 mg/kg MMC. Control mice (PBS) were sacrificed when MMC-injected mice reached the humane endpoint. Scale bar, 2.5 mm. In each image, a relevant area is shown at higher magnification (10x).


E . IF detection of FANCD2 foci in WT and PRIMPOL KO cells 48 h after TMP - UVA ( 2 µM TMP , 2 h followed by 5 s irradiation ) with or without MCM3 downregulation . Nuclear DNA is counterstained with DAPI . Scale bar , 25 µm .

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

E. IF detection of FANCD2 foci in WT and PRIMPOL KO cells 48 h after TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation) with or without MCM3 downregulation. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.


G . Cell survival assays of WT and PRIMPOL KO cells with or without MCM3 downregulation in the presence of increasing amounts of MMC or TMP ( followed by 90 sec UVA irradiation ) for 72 h . The same control curves without MCM3 downregulation are also shown as part of Figure EV6D . Each point in the curve represents the percentage of surviving cells ( average and SD of three assays for MMC and two assays for TMP ) . Statistical analysis was conducted with two - way ANOVA followed by Bonferroni post - test . p - values of comparisons between KO and KO - siMCM3 are indicated .

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

G. Cell survival assays of WT and PRIMPOL KO cells with or without MCM3 downregulation in the presence of increasing amounts of MMC or TMP (followed by 90 sec UVA irradiation) for 72 h. The same control curves without MCM3 downregulation are also shown as part of Figure EV6D. Each point in the curve represents the percentage of surviving cells (average and SD of three assays for MMC and two assays for TMP). Statistical analysis was conducted with two-way ANOVA followed by Bonferroni post-test. p-values of comparisons between KO and KO-siMCM3 are indicated.


Montages of representative DAPI - stained nuclei are shown for gene knockdowns that led to reduced nuclear size . Cell - to - cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability . Ten nuclei per column , organized by maximum nuclear size z - score . Scale bar , 25 µm . Median nuclear size z - scores are plotted for gene knockdowns that led to reduced nuclear size . Data are based on three different siRNA oligo sequences for each gene and two biological replicates . Error bars represent the SEM for biological replicates .

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

Montages of representative DAPI-stained nuclei are shown for gene knockdowns that led to reduced nuclear size. Cell-to-cell variability in the amount of ELYS knockdown likely accounts for apparent nuclear size variability. Ten nuclei per column, organized by maximum nuclear size z-score. Scale bar, 25 µm. Median nuclear size z-scores are plotted for gene knockdowns that led to reduced nuclear size. Data are based on three different siRNA oligo sequences for each gene and two biological replicates. Error bars represent the SEM for biological replicates.


MCF - 10AT1k . cl2 cells were transfected with control or ELYS siRNA . ( A ) Representative images of cells stained for lamin B2 and B1 are shown . ( B ) Representative images of cells stained for lamin A / C and B1 are shown . ( C ) For each experiment , nuclear lamin B1 , B2 , and A / C staining intensities were quantified for 44 - 159 nuclei per condition ( 96 nuclei on average ) and normalized to the negative control . Three biological replicates , data from one representative experiment shown . ( D ) Representative images of cells stained for FG - Nups ( mAb414 ) and lamin B1 are shown . ( E ) Representative images of cells stained for Nup133 and lamin B1 are shown . ( F ) For each experiment , nuclear mAb414 and Nup133 staining intensities were quantified for 31 - 83 nuclei per condition ( 60 nuclei on average ) and normalized to the negative control . To measure NPC densities , confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei . NPC numbers were counted per unit area for 18 - 50 nuclei per condition ( 30 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. (A) Representative images of cells stained for lamin B2 and B1 are shown. (B) Representative images of cells stained for lamin A/C and B1 are shown. (C) For each experiment, nuclear lamin B1, B2, and A/C staining intensities were quantified for 44-159 nuclei per condition (96 nuclei on average) and normalized to the negative control. Three biological replicates, data from one representative experiment shown. (D) Representative images of cells stained for FG-Nups (mAb414) and lamin B1 are shown. (E) Representative images of cells stained for Nup133 and lamin B1 are shown. (F) For each experiment, nuclear mAb414 and Nup133 staining intensities were quantified for 31-83 nuclei per condition (60 nuclei on average) and normalized to the negative control. To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 18-50 nuclei per condition (30 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were transfected with control or ELYS siRNA . Representative images of cells stained for Ran and lamin B1 are shown . ( H ) Representative images of cells stained for NTF2 and lamin A / C are shown . ( I ) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control . To measure the N / C staining intensity ratio of a given cell , the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region . For each experiment , 54 - 216 nuclei were quantified per condition ( 112 nuclei on average ) . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were transfected with control or ELYS siRNA. Representative images of cells stained for Ran and lamin B1 are shown. (H) Representative images of cells stained for NTF2 and lamin A/C are shown. (I) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the negative control. To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region. For each experiment, 54-216 nuclei were quantified per condition (112 nuclei on average). Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were co - transfected with control or ELYS siRNA and plasmids expressing eGFP alone or siRNA - resistant eGFP - ELYS . Cell lysates were analyzed by western blot and probed for ELYS and tubulin . ELYS band intensity was normalized to tubulin and then normalized to the eGFP / negative siRNA control . ELYS concentrations are indicated in the row labeled [ ELYS ] ( average ± SD ) . Quantification was performed on two biological replicates . Cells were stained with an antibody against lamin B1 . Representative images are shown . ( B ) For each experiment , nuclear cross - sectional areas were quantified for 68 - 298 nuclei per condition ( 162 nuclei on average ) and averaged . Three biological replicates , data from one representative experiment shown . ( C ) For each experiment , nuclear lamin B2 staining intensities were quantified for 29 - 81 nuclei per condition ( 60 nuclei on average ) and normalized to the eGFP / negative siRNA control . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing eGFP alone or siRNA-resistant eGFP-ELYS. Cell lysates were analyzed by western blot and probed for ELYS and tubulin. ELYS band intensity was normalized to tubulin and then normalized to the eGFP/negative siRNA control. ELYS concentrations are indicated in the row labeled [ELYS] (average ± SD). Quantification was performed on two biological replicates. Cells were stained with an antibody against lamin B1. Representative images are shown. (B) For each experiment, nuclear cross-sectional areas were quantified for 68-298 nuclei per condition (162 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown. (C) For each experiment, nuclear lamin B2 staining intensities were quantified for 29-81 nuclei per condition (60 nuclei on average) and normalized to the eGFP/negative siRNA control. Two biological replicates, data from one representative experiment shown.


Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 58 - 299 cells were examined per condition . Three biological replicates , data from one representative experiment shown .

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

Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 58-299 cells were examined per condition. Three biological replicates, data from one representative experiment shown.


Cells were stained with antibodies against Ran and NTF2 . For each experiment , 62 - 130 nuclei were quantified per condition ( 92 nuclei on average ) . Two biological replicates , data from one representative experiment shown . ( E ) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the eGFP control . ( F ) To measure the N / C staining intensity ratio of a given cell , the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region .

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

Cells were stained with antibodies against Ran and NTF2. For each experiment, 62-130 nuclei were quantified per condition (92 nuclei on average). Two biological replicates, data from one representative experiment shown. (E) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the eGFP control. (F) To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region.


Transfections were performed as in ( A ) and cells were stained with an antibody against FG - Nups ( mAb414 ) and Nup133 . Representative images for mAb414 staining are shown . Confocal imaging was performed through nuclear cross - sections as well as on the surface of nuclei . ( H ) To measure NPC densities , confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei . NPC numbers were counted per unit area for 25 - 116 nuclei per condition ( 53 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

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

Transfections were performed as in (A) and cells were stained with an antibody against FG-Nups (mAb414) and Nup133. Representative images for mAb414 staining are shown. Confocal imaging was performed through nuclear cross-sections as well as on the surface of nuclei. (H) To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 25-116 nuclei per condition (53 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were co - transfected with control or ELYS siRNA and plasmids expressing mCherry alone or mCherry - importin α . Cells were stained with an antibody against lamin B2 . Representative images are shown . Scale bar , 10 µm . ( B ) For each experiment , nuclear cross - sectional areas were quantified for 30 - 172 nuclei per condition ( 84 nuclei on average ) and averaged . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing mCherry alone or mCherry-importin α. Cells were stained with an antibody against lamin B2. Representative images are shown. Scale bar, 10 µm. (B) For each experiment, nuclear cross-sectional areas were quantified for 30-172 nuclei per condition (84 nuclei on average) and averaged. Two biological replicates, data from one representative experiment shown.


Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 74 - 218 cells were examined per condition . Two biological replicates , data from one representative experiment shown .

Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 74-218 cells were examined per condition. Two biological replicates, data from one representative experiment shown.


For each experiment , nuclear lamin B2 staining intensities were quantified for 34 - 187 nuclei per condition ( 85 nuclei on average ) and normalized to the mCherry / negative siRNA control . To measure bulk import capacity for importin α / β cargos , cells were co - transfected as in ( A ) along with a plasmid expressing GFP - 3x SV40 NLS . For each experiment , nuclear GFP - NLS intensities were quantified for 22 - 188 nuclei per condition ( 73 nuclei on average ) and normalized to the mCherry / negative siRNA control . Two biological replicates , data from one representative experiment shown .

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

For each experiment, nuclear lamin B2 staining intensities were quantified for 34-187 nuclei per condition (85 nuclei on average) and normalized to the mCherry/negative siRNA control. To measure bulk import capacity for importin α/β cargos, cells were co-transfected as in (A) along with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 22-188 nuclei per condition (73 nuclei on average) and normalized to the mCherry/negative siRNA control. Two biological replicates, data from one representative experiment shown.


MCF - 10AT1k . cl2 cells were transfected with plasmids expressing mCherry alone or mCherry - RanQ69L and stained with an antibody against lamin B2 . Representative images are shown .

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

MCF-10AT1k.cl2 cells were transfected with plasmids expressing mCherry alone or mCherry-RanQ69L and stained with an antibody against lamin B2. Representative images are shown.


MCF - 10AT1k . cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1 . Representative images are shown .

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

MCF-10AT1k.cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1. Representative images are shown.


For each experiment , nuclear cross - sectional areas were quantified for 32 - 271 nuclei per condition ( 123 nuclei on average ) and averaged . Three biological replicates , data from one representative experiment shown .

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

For each experiment, nuclear cross-sectional areas were quantified for 32-271 nuclei per condition (123 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown.


To measure bulk import capacity for importin α / β cargos , experiments were performed as in ( A - B ) except that cells were also transfected with a plasmid expressing GFP - 3x SV40 NLS . For each experiment , nuclear GFP - NLS intensities were quantified for 11 - 75 nuclei per condition ( 35 nuclei on average ) and normalized to the appropriate control . Two biological replicates , data from one representative experiment shown .

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

To measure bulk import capacity for importin α/β cargos, experiments were performed as in (A-B) except that cells were also transfected with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 11-75 nuclei per condition (35 nuclei on average) and normalized to the appropriate control. Two biological replicates, data from one representative experiment shown.


Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated . For each experiment , 31 - 130 cells were examined per condition . Three biological replicates , data from one representative experiment shown .

Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 31-130 cells were examined per condition. Three biological replicates, data from one representative experiment shown.


Cell lysates from control and XPO1 siRNA transfected MCF - 10AT1k . cl2 cells were analyzed by western blot and probed for XPO1 and tubulin . One representative western blot is shown . XPO1 band intensity was normalized to tubulin . Quantification from two biological replicates is shown .

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

Cell lysates from control and XPO1 siRNA transfected MCF-10AT1k.cl2 cells were analyzed by western blot and probed for XPO1 and tubulin. One representative western blot is shown. XPO1 band intensity was normalized to tubulin. Quantification from two biological replicates is shown.


MCF - 10AT1k . cl2 cells were transfected with control or XPO1 siRNA . Lamin B1 and lamin A / C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates . For each experiment , 108 - 865 nuclei were quantified per condition ( 416 nuclei on average ) . Average and SD values based on averages from these four experiments are plotted , normalized to the negative siRNA control .

MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. Lamin B1 and lamin A/C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates. For each experiment, 108-865 nuclei were quantified per condition (416 nuclei on average). Average and SD values based on averages from these four experiments are plotted, normalized to the negative siRNA control.


MCF - 10AT1k . cl2 cells were transfected with the indicated siRNAs and nuclear cross - sectional areas were quantified . For each experiment , 203 - 604 nuclei were quantified per condition ( 403 nuclei on average ) . Average and SD values based on averages from four biological replicates are plotted , normalized to the negative siRNA control .

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

MCF-10AT1k.cl2 cells were transfected with the indicated siRNAs and nuclear cross-sectional areas were quantified. For each experiment, 203-604 nuclei were quantified per condition (403 nuclei on average). Average and SD values based on averages from four biological replicates are plotted, normalized to the negative siRNA control.


MCF - 10AT1k . cl2 cells were transfected with control siRNA or with siRNA against XPO1 . Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high - throughput imaging as previously described [ 123 ] ( see Methods ) . The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates .

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

MCF-10AT1k.cl2 cells were transfected with control siRNA or with siRNA against XPO1. Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high-throughput imaging as previously described [123] (see Methods). The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates.


MCF - 10AT1k . cl2 cells were transfected with control or XPO1 siRNA . For the bottom row of images , cells transfected with control siRNA were treated with 20 ng / ml leptomycin B for 24 hours . Cells were fixed and stained with 2 µg / ml FITC . Representative images are shown . Scale bar , 20 µm . For each experiment , nuclear cross - sectional areas and nuclear FITC staining intensities were quantified for 105 - 620 nuclei per condition ( 267 nuclei on average ) and normalized to the negative control . Two biological replicates , data from one representative experiment shown .

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

MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. For the bottom row of images, cells transfected with control siRNA were treated with 20 ng/ml leptomycin B for 24 hours. Cells were fixed and stained with 2 µg/ml FITC. Representative images are shown. Scale bar, 20 µm. For each experiment, nuclear cross-sectional areas and nuclear FITC staining intensities were quantified for 105-620 nuclei per condition (267 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.


( 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 .

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

(A 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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.)


( A ) Immunoblot analysis for BIK1 - HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min . Arrowheads indicate phosphorylated BIK1 - HA .

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

(A) Immunoblot analysis 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 ) .

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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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.)


( 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 .

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

(A) 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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C 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.)


( 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 .

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

(A) 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 .

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

(C, 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.


( 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 . )

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

(A) 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 . )

(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 . )

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

(C) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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.


( 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 .

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(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 .

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(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 .

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. (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 .

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(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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) 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.


( 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 . )

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

(A) 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 . )

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

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

(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 . )

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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.)


( 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 . )

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

(A) 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 .

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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.


A . GFP - Snc1 - PEM protein accumulates in atg1Δ , atg8Δ , and atg11Δ mutant cells . Lysates were prepared from wild type ( WT ) , ypt1 - 1 ( for comparison ) , atg1Δ , atg8Δ , and atg11Δ mutant cells transformed with a 2μ plasmid expressing GFP - Snc1 - PEM from the TPI promoter . The level of GFP - Snc1 - PEM was determined using immunoblot analysis with anti - GFP antibodies . The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control .

A. GFP-Snc1-PEM protein accumulates in atg1Δ, atg8Δ, and atg11Δ mutant cells. Lysates were prepared from wild type (WT), ypt1-1 (for comparison), atg1Δ, atg8Δ, and atg11Δ mutant cells transformed with a 2μ plasmid expressing GFP-Snc1-PEM from the TPI promoter. The level of GFP-Snc1-PEM was determined using immunoblot analysis with anti-GFP antibodies. The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control.