Figure 4Detection 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 ) . 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 .

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

Figure 4Detection 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).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.


Figure 5Relative 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Relative 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.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).


Figure 6Hypocotyl 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Hypocotyl 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).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).


Figure 1D . 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 ) .

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Figure 1D. 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).


Figure 2B . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. 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.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.


Figure 3H . 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 .

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Figure 3H. 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.


Figure 4C . 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 .

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Figure 4C. 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.


Figure 5A . 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 .

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Figure 5A. 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.


Figure 6A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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).


Figure 7E . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7E. 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.


Figure 1Montages 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 .

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Figure 1Montages 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.


Figure 2MCF - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2MCF-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.


Figure 3MCF - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3MCF-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.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.


Figure 4MCF - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4MCF-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.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.


Figure 5MCF - 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 . 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 . 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 .

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

Figure 5MCF-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.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.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.


Figure 6Cell 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 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Cell 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 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.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.


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


Fig 1A . 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 . B . GFP - Snc1 - PEM protein accumulates in aberrant ER structures in atg1Δ , atg8Δ , and atg11Δ mutant cells . The ER - marker Sec61 was tagged with mCherry in strains from panel A , and the cells were examined by live - cell microscopy . Shown from left to right : DIC , GFP , mCherry , merge , % cells with intracellular GFP - Snc1 - PEM ( number of cells with internal GFP - Snc1 - PEM / number of cells visualized ) , and % cells in which intra - cellular Snc1 - PEM co - localizes with Sec61 . C . UPR is induced in atg1Δ , atg8Δ , and atg11Δ mutant cells . Cells from panel A were transformed with a second plasmid that expresses β - gal from a UPR promoter . UPR was determined and expressed as % of the WT response . D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level ( DD - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level ( D ) , intra - cellular accumulation of GFP - Snc1 - PEM , ( E ) , D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level ( D ) , intra - cellular accumulation of GFP - Snc1 - PEM , ( E ) , and induction of the UPR response ( F ) . Wild type ( WT ) , atg19Δ , atg11Δ , atg32Δ , and atg36Δ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for panels A - C , respectively . E . Shown from left to right : DIC , GFP , and % cells with intracellular Snc1 - PEM structures . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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

Fig 1A. 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.B. GFP-Snc1-PEM protein accumulates in aberrant ER structures in atg1Δ, atg8Δ, and atg11Δ mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61.C. UPR is induced in atg1Δ, atg8Δ, and atg11Δ mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response.D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (DD-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E),D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E), and induction of the UPR response (F). Wild type (WT), atg19Δ, atg11Δ, atg32Δ, and atg36Δ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. E. Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


Fig 2A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) , A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B ) A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B ) , and induction of the UPR response ( C , atg1 ∆ is shown as a positive control ) . D - G . Atg9 is epistatic to Atg11 in macro - ER - phagy . ATG9 was deleted in wild type and atg11 ∆ mutant cells and the effects of overexpression of GFP - Snc1 - PEM were determined in the single and double mutants as described in Fig 1 legend . D . Deletion of ATG9 in wild type ( WT ) or atg11 ∆ mutant cells results in an increase of GFP - Snc1 - PEM protein level similar to the increase in atg11 ∆ mutant cells . E . Deletion of ATG9 in wild type or atg11 ∆ mutant cells results in an increase of intracellular GFP - Snc1 - PEM fluorescence . However , only ~ 20 % of the atg9 ∆ single - , and atg9 ∆ atg11 ∆ double - mutant cells accumulate GFP - Snc1 - PEM in aberrant structures , as compared with ~ 75 % of atg11 ∆ mutant cells . Shown from top to bottom : DIC , GFP , % cells with aberrant intracellular GFP - Snc1 - PEM structures , ratio of GFP - Snc1 - PEM fluorescence inside / PM ( 30 cells were analyzed for each strain ) . F . UPR is induced in atg11 ∆ , but not in atg9 ∆ single - and atg9 ∆ atg11 ∆ double - mutant cells overexpressing GFP - Snc1 - PEM . G . UPR can be induced in atg9 ∆ single - , and atg9 atg11 ∆ double - mutant cells overexpressing GFP - Snc1 - PEM by tunicamycin . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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

Fig 2A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A),A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B)A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B), and induction of the UPR response (C,atg1∆ is shown as a positive control).D-G. Atg9 is epistatic to Atg11 in macro-ER-phagy. ATG9 was deleted in wild type and atg11∆ mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in Fig 1 legend. D. Deletion of ATG9 in wild type (WT) or atg11∆ mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in atg11∆ mutant cells.E. Deletion of ATG9 in wild type or atg11∆ mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the atg9∆ single-, and atg9∆ atg11∆ double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of atg11∆ mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain).F. UPR is induced in atg11∆, but not in atg9∆ single- and atg9∆ atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM.G. UPR can be induced in atg9∆ single-, and atg9 atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


Fig 3A - C . ATG9 was deleted in ypt1 - 1 , WT and sec12ts mutant cells and the effect of overexpression of GFP - Snc1 - PEM was determined in single and double mutant cells . Experiments were performed as described for Fig 1A - 1C , respectively . A . Snc1 - PEM is increased ~ 3 fold in atg9 ∆ and sec12ts as compared to ~ 20 fold in ypt1 - 1 mutant cells . Importantly , atg9 ∆ is epstatic to the ypt1 - 1 , but not to the sec12ts , mutation . Left , immuno - blot analysis , increase of the protein level in mutant versus the WT cells is shown under the blot ; right , a bar graph summarizing the quantified data . Whereas deletion of ATG9 in ypt1 - 1 mutant cells results in three fold lower accumulation of GFP - Snc1 - PEM in aberrant structures ( 85 % to 27 % ) , its deletion in sec12ts mutant cells results in two fold increased accumulation ( ~ 40 % to ~ 80 % ) . Shown from top to bottom : DIC , GFP , and % cells with intracellular Snc1 - PEM structures . C . Deletion of ATG9 in ypt1 - 1 mutant cells overexpressing Snc1 - PEM results in ~ 3 . 5 lower UPR ( p - value < 0 . 0005 ) , but a slightly increased UPR in sec12ts mutant cells ( p - value = 0 . 05 ) . D . Atg9 is present on aberrant ER structures that accumulate in ypt1 - 1 mutant cells . WT and ypt1 - 1 mutant cells expressing Atg9 - mCherry from the chromosome and GFP - Snc1 - PEM from a 2μ plasmid were analyzed by live - cell microscopy . Whereas in WT cells ( top ) , the GFP - Snc1 - PEM localizes to the cell membrane and Atg9 - mCherry to intracellular puncta , the two proteins co - localize in 100 % of the ypt1 - 1 mutant cells ( bottom ) that accumulate intracellular GFP - Snc1 - PEM structures ( ~ 80 % ) . Shown from left to right : DIC , GFP , mCherry , merge , % cells with intracellular Snc1 - PEM , and % cells with co - localization ( number of cells with observed phenotype / total number of cells analyzed ) . E . GFP - Snc1 - PEM is delivered to the vacuole for degradation in sec12ts mutant cells . Accumulation of GFP - Snc1 - PEM in vacuoles of sec12ts mutant cells , with and without deletion of the vacuolar protease Pep4 , was determined using the FM4 - 64 dye , which labels the vacuolar membrane . Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP - Snc1 - PEM accumulates inside the vacuole ( from 8 % to 100 % ) . Shown from left to right : DIC , GFP , FM4 - 64 ( vacuolar membrane ) , merge , % cells with aberrant GFP - Snc1 - PEM structures , % cells with GFP - Snc1 - PEM outside vacuole , and % cells with GFP - Snc1 - PEM inside the vacuole . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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

Fig 3A-C.ATG9 was deleted in ypt1-1, WT and sec12ts mutant cells and the effect of overexpression of GFP-Snc1-PEM was determined in single and double mutant cells. Experiments were performed as described for Fig 1A-1C, respectively. A. Snc1-PEM is increased ~3 fold in atg9∆ and sec12ts as compared to ~20 fold in ypt1-1 mutant cells. Importantly, atg9∆ is epstatic to the ypt1-1, but not to the sec12ts, mutation. Left, immuno-blot analysis, increase of the protein level in mutant versus the WT cells is shown under the blot; right, a bar graph summarizing the quantified data. Whereas deletion of ATG9 in ypt1-1 mutant cells results in three fold lower accumulation of GFP-Snc1-PEM in aberrant structures (85% to 27%), its deletion in sec12ts mutant cells results in two fold increased accumulation (~40% to ~80%). Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.C. Deletion of ATG9 in ypt1-1 mutant cells overexpressing Snc1-PEM results in ~3.5 lower UPR (p-value<0.0005), but a slightly increased UPR in sec12ts mutant cells (p-value = 0.05).D. Atg9 is present on aberrant ER structures that accumulate in ypt1-1 mutant cells. WT and ypt1-1 mutant cells expressing Atg9-mCherry from the chromosome and GFP-Snc1-PEM from a 2μ plasmid were analyzed by live-cell microscopy. Whereas in WT cells (top), the GFP-Snc1-PEM localizes to the cell membrane and Atg9-mCherry to intracellular puncta, the two proteins co-localize in 100% of the ypt1-1 mutant cells (bottom) that accumulate intracellular GFP-Snc1-PEM structures (~80%). Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular Snc1-PEM, and % cells with co-localization (number of cells with observed phenotype / total number of cells analyzed).E. GFP-Snc1-PEM is delivered to the vacuole for degradation in sec12ts mutant cells. Accumulation of GFP-Snc1-PEM in vacuoles of sec12ts mutant cells, with and without deletion of the vacuolar protease Pep4, was determined using the FM4-64 dye, which labels the vacuolar membrane. Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP-Snc1-PEM accumulates inside the vacuole (from 8% to 100%). Shown from left to right: DIC, GFP, FM4-64 (vacuolar membrane), merge, % cells with aberrant GFP-Snc1-PEM structures, % cells with GFP-Snc1-PEM outside vacuole, and % cells with GFP-Snc1-PEM inside the vacuole. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


Fig 4A . The ypt1 - 1 mutation is epistatic to vps21 ∆ in ER - phagy . yDsRed - Snc1 - PEM was overexpressed in WT , vps21 ∆ , ypt1 - 1 and ypt1 - 1vps21 ∆ double mutant cells that also expressed the autophagosomal marker yEGFP - Atg8 . Cells were analyzed by live - cell microscopy . Shown from left to right : DIC , DsRed , GFP , merge , % cells Atg8 dots , number of Atg8 dots per cell , and % cells in which the Atg8 dots co - localize with Snc1 - PEM . About 50 % of WT and 85 % of vps21 ∆ mutant cells contain ~ 1 dot of Atg8 representing the AP . Importantly , in ~ 70 % of the vps21 ∆ mutant cells Snc1 - PEM co - localizes with the APs , as compared to ~ 4 % in WT cells . In contrast , ~ 90 % ypt1 - 1 and ypt1 - 1vps21 ∆ mutant cells contain three APs per cell , and Snc1 - PEM does not co - localize with them . Arrows point to co - localization ; arrowheads point to either Atg8 dots or GFP - Snc1 - PEM that do not co - localize . The following effects of overexpression of GFP - Snc1 - PEM in WT and ypt1 - 1 mutant cells , without and with ire1Δ or hac1Δ , were analyzed as described for Fig 1A - 1C , respectively : protein level ( B ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( C ) , and UPR induction ( D ) . B . Deletion of either Ire1 or Hac1 does not affect the level of Snc1 - PEM accumulation in WT or ypt1 - 1 mutant cells . C . Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1 - 1 mutant cells that accumulate aberrant intra - cellular Snc1 - PEM . Shown from top to bottom : DIC , GFP , and % cells with intracellular Snc1 - PEM structures . D . Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1 - 1 mutant cells . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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

Fig 4A. The ypt1-1 mutation is epistatic to vps21∆ in ER-phagy. yDsRed-Snc1-PEM was overexpressed in WT, vps21∆, ypt1-1 and ypt1-1vps21∆ double mutant cells that also expressed the autophagosomal marker yEGFP-Atg8. Cells were analyzed by live-cell microscopy. Shown from left to right: DIC, DsRed, GFP, merge, % cells Atg8 dots, number of Atg8 dots per cell, and % cells in which the Atg8 dots co-localize with Snc1-PEM. About 50% of WT and 85% of vps21∆ mutant cells contain ~1 dot of Atg8 representing the AP. Importantly, in ~70% of the vps21∆ mutant cells Snc1-PEM co-localizes with the APs, as compared to ~4% in WT cells. In contrast, ~90% ypt1-1 and ypt1-1vps21∆ mutant cells contain three APs per cell, and Snc1-PEM does not co-localize with them. Arrows point to co-localization; arrowheads point to either Atg8 dots or GFP-Snc1-PEM that do not co-localize.The following effects of overexpression of GFP-Snc1-PEM in WT and ypt1-1 mutant cells, without and with ire1Δ or hac1Δ, were analyzed as described for Fig 1A-1C, respectively: protein level (B), accumulation of GFP-Snc1-PEM in aberrant structures (C), and UPR induction (D). B. Deletion of either Ire1 or Hac1 does not affect the level of Snc1-PEM accumulation in WT or ypt1-1 mutant cells.C. Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1-1 mutant cells that accumulate aberrant intra-cellular Snc1-PEM. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.D. Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1-1 mutant cells. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.