Figure 7Representative nuclease assays with MRE11 , MR , MRN and pCtIP , as indicated , on 5 ' - end labeled dsDNA with a single streptavidin - blocked end ( S ) . The substrate contained 5 phosphorothioate bonds ( PTO ) at both 3 ' - ends to prevent exonucleolytic DNA degradation . Reactions were incubated for 2 h . Representative nuclease assays as in ( A ) , but with a substrate containing a nick in the top oligonucleotide ( 20 nt away from the 3 ' - end , see cartoon ) . Quantitation of experiments such as shown in ( A ) , with pCtIP . Averages shown ; n ≥ 4 ; error bars , SEM . Quantitation of experiments such as shown in ( B ) , with and without pCtIP . Averages shown ; n = 3 ; error bars , SEM .

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Figure 7Representative nuclease assays with MRE11, MR, MRN and pCtIP, as indicated, on 5'-end labeled dsDNA with a single streptavidin-blocked end (S). The substrate contained 5 phosphorothioate bonds (PTO) at both 3'-ends to prevent exonucleolytic DNA degradation. Reactions were incubated for 2 h.Representative nuclease assays as in (A), but with a substrate containing a nick in the top oligonucleotide (20 nt away from the 3'-end, see cartoon).Quantitation of experiments such as shown in (A), with pCtIP. Averages shown; n ≥ 4; error bars, SEM.Quantitation of experiments such as shown in (B), with and without pCtIP. Averages shown; n = 3; error bars, SEM.


Figure 1A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup / Cdt1 RNAi , cyclin E RNAi , Cdk2 RNAi , Dacapo overexpression ( + Dacapo ) , and 1 mM HU . B Analysis of Mcm2 ‐ 7 nuclear chromatin association at different points in the cell cycle . Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2 ‐ 7 by Western blot . A non ‐ specific band is indicated by an asterisk ( * ) . C Quantification of the ratio of chromatin ‐ bound Mcm2 ‐ 7 relative to Orc2 ( log10 scale ) for a minimum of 11 replicates ( mean ± SD ) .

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Figure 1A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.B Analysis of Mcm2‐7 nuclear chromatin association at different points in the cell cycle. Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2‐7 by Western blot. A non‐specific band is indicated by an asterisk (*).C Quantification of the ratio of chromatin‐bound Mcm2‐7 relative to Orc2 (log10 scale) for a minimum of 11 replicates (mean ± SD).


Figure 2Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h ( top ) . Western blot analysis of Mcm2 ‐ 7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2 ‐ 7 , Orc2 , and Dacapo .

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Figure 2Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h (top). Western blot analysis of Mcm2‐7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2‐7, Orc2, and Dacapo.


Figure 3B FACS profiles of DNA content for each cell population assayed . C Western blot analysis for Orc2 and Mcm2 ‐ 7 in the nuclear chromatin fraction and Orc2 , Mcm2 ‐ 7 , Dup / Cdt1 , and cyclin E in the cytoplasmic fraction for each condition assayed .

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Figure 3B FACS profiles of DNA content for each cell population assayed.C Western blot analysis for Orc2 and Mcm2‐7 in the nuclear chromatin fraction and Orc2, Mcm2‐7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.


Figure 4A Genome ‐ wide analysis of ORC localization by ChIP ‐ chip . ORC enrichment from asynchronous cells is depicted for a 5 ‐ Mb section of chromosome 2L . B Genome ‐ wide analysis of Mcm2 ‐ 7 localization in early G1 by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from cyclin E RNAi ‐ depleted cells is depicted for a 5 ‐ Mb section of chromosome 2L . C Venn diagram depicting the overlap between ORC and Mcm2 ‐ 7 peaks .

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Figure 4A Genome‐wide analysis of ORC localization by ChIP‐chip. ORC enrichment from asynchronous cells is depicted for a 5‐Mb section of chromosome 2L.B Genome‐wide analysis of Mcm2‐7 localization in early G1 by ChIP‐chip. Mcm2‐7 enrichment from cyclin E RNAi‐depleted cells is depicted for a 5‐Mb section of chromosome 2L.C Venn diagram depicting the overlap between ORC and Mcm2‐7 peaks.


Figure 5A Genome ‐ wide analysis of Mcm2 ‐ 7 localization at the G1 / S transition by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from HU ‐ arrested cells is depicted for a 5 ‐ Mb section of chromosome 2L . Inset : transcribed ( green ) and non ‐ transcribed ( red ) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom . B Bimodal distribution of Mcm2 ‐ 7 enrichment over transcribed and non ‐ transcribed genes . Histogram showing the distribution of probe scores found within transcribed ( green ) and non ‐ transcribed ( red ) genes . C " Meta " ‐ gene analysis of Mcm2 ‐ 7 enrichment for different deciles of gene expression and their aggregated probe intensities . D Mcm2 ‐ 7 is displaced from chromatin by DNA replication . Genome ‐ wide analysis of Mcm2 ‐ 7 localization in late S ‐ phase ( 6 h post HU release ) by ChIP ‐ chip . Mcm2 ‐ 7 enrichment is depicted for a 10 ‐ Mb section of chromosome 2L ( filled gray ) , replication timing profile ( black line ) and early ( yellow ) and late ( purple ) replication timing domains . E Box ‐ plots representing late S ‐ phase Mcm2 ‐ 7 ChIP signal found within early ( 246 ) or late ( 167 ) replication domains .

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Figure 5A Genome‐wide analysis of Mcm2‐7 localization at the G1/S transition by ChIP‐chip. Mcm2‐7 enrichment from HU‐arrested cells is depicted for a 5‐Mb section of chromosome 2L. Inset: transcribed (green) and non‐transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.B Bimodal distribution of Mcm2‐7 enrichment over transcribed and non‐transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non‐transcribed (red) genes.C "Meta"‐gene analysis of Mcm2‐7 enrichment for different deciles of gene expression and their aggregated probe intensities.D Mcm2‐7 is displaced from chromatin by DNA replication. Genome‐wide analysis of Mcm2‐7 localization in late S‐phase (6 h post HU release) by ChIP‐chip. Mcm2‐7 enrichment is depicted for a 10‐Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.E Box‐plots representing late S‐phase Mcm2‐7 ChIP signal found within early (246) or late (167) replication domains.


Figure 1LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) and cell lysates ( Lysate ) were collected and immunoblotted with the indicated antibodies （ A ） . LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected and ( B - E ) IL - 1β secretion ( B ) , IL - 18 secretion ( C ) and TNF - ɑ secretion ( D ) and LDH release ( E ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments . LPS - primed WT and Vdr - / - BMDMs were infected with 50 MOI S . Typhimurium for 2 h . ( F ) HBVLV - Vdr expression in Vdr - / - BMDMs by lentivirus - mediated transduction primed with LPS and infected with 50 MOI S . Typhimurium for 2 h , cell lysates , and culture supernatants ( SN ) were collected and immunoblotted with the indicated antibodies . ( G ) LPS - primed WT and Vdr - / - BMDMs were treated with 1 µg / ml FLIC ( LFn - Flagellin and PA ) or PrgJ （ 1 µg / ml LFn - PrgJ and PA ） for 1 h , total mixtures ( culture supernatants and cell lysates ) were collected and immunoblotted with the indicated antibodies . ( H ) LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) and cell lysates were collected and immunoblotted with the indicated antibodies . LPS - primed WT , Vdr - / - , NLRC4 - / - and Vdr - / - NLRC4 - / - BMDMs were treated with FLIC for 1 h , Culture supernatants ( SN ) were collected and ( I - L ) IL - 1β secretion ( I ) , IL - 18 secretion ( J ) and TNF - ɑ secretion ( K ) and LDH release ( L ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 In each panel , data are representative of at least three independent experiments .

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Figure 1LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) and cell lysates(Lysate) were collected and immunoblotted with the indicated antibodies（A）.LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) were collected and (B-E) IL-1β secretion(B), IL-18 secretion (C) and TNF-ɑ secretion (D) and LDH release(E) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. (F) HBVLV-Vdr expression in Vdr-/- BMDMs by lentivirus-mediated transduction primed with LPS and infected with 50 MOI S. Typhimurium for 2 h, cell lysates, and culture supernatants (SN) were collected and immunoblotted with the indicated antibodies.(G) LPS-primed WT and Vdr-/- BMDMs were treated with 1 µg/ml FLIC (LFn-Flagellin and PA) or PrgJ （1 µg/ml LFn- PrgJ and PA）for 1 h, total mixtures( culture supernatants and cell lysates) were collected and immunoblotted with the indicated antibodies.(H) LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) and cell lysates were collected and immunoblotted with the indicated antibodies.LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) were collected and (I-L) IL-1β secretion(I), IL-18 secretion (J) and TNF-ɑ secretion (K) and LDH release(L) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.


Figure 3LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) and Cell lysates were collected and immunoblotted with the indicated antibodies ( A ) . ELISALPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with different MOI ( 0 , 10 , 50 ) S . Typhimurium for 2 h . Culture supernatants ( SN ) were collected antibodies ( A ) . ELISA detected TNF - ɑ ( B ) andIL - 1β ( C ) in supernatants . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 . LPS - primed WT , Vdr - / - , NLRP3 - / - and NLRP3 - / - Vdr - / - BMDMs were infected with S . Typhimurium Bacterial burden was detected in spleens ( D ) , PCF ( E ) , and blood ( F ) of WT , Vdr - / - , NLRP3 - / - , and NLRP3 - / - Vdr - / - mice 24 h after S . Typhimurium infection . Data information : n ≥ 3 biological replicates . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 .

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Figure 3LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) and Cell lysates were collected and immunoblotted with the indicated antibodies(A).ELISALPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) were collected antibodies(A).ELISA detected TNF-ɑ(B)andIL-1β (C) in supernatants. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with S. Typhimurium Bacterial burden was detected in spleens(D), PCF(E), and blood (F)of WT, Vdr-/-, NLRP3-/-, and NLRP3-/- Vdr-/- mice 24 h after S. Typhimurium infection. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.


Figure 4A LPS - primed WT and Vdr - / - BMDMs were infected with S . Typhimurium at an MOI of 50 for 2 h . Endogenous ASC specks ( arrows ) were represented and quantified by immunofluorescence images . The data show representative results from three independent experiments : scale bar , 20 μm . Data are presented as the mean ± SEM ; determined by Student ' s t - test ; * * * , p < 0 . 001 . B ASC oligomerization induced by the indicated stimuli in WT and Vdr − / − BMDMs primed with LPS .

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Figure 4A LPS-primed WT and Vdr-/- BMDMs were infected with S. Typhimurium at an MOI of 50 for 2 h. Endogenous ASC specks (arrows) were represented and quantified by immunofluorescence images. The data show representative results from three independent experiments: scale bar, 20 μm. Data are presented as the mean ± SEM; determined by Student's t-test ; ***,p < 0.001.B ASC oligomerization induced by the indicated stimuli in WT and Vdr −/− BMDMs primed with LPS.


Figure 5LPS - primed BMDMs were infected with S . Typhimurium at an MOI of 50 for 2h . Lysates were immunoprecipitated ( IP ) using an anti - VDR antibody and were immunoblotted with the indicated antibodies ( A ) . Whole - cell lysates were shown as the input . Data are representative of three independent experiments . ( B - C ) Flag - VDR was co - expressed with Myc - NLRC4 in HEK293T cells ; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies . Whole - cell lysates were shown as the input . Data are representative of three independent experiments . ( D ) Purified GST - VDR was incubated with purified His - NLRC4 for 2 h . His - NLRC4 - bound to GST - VDR was pulled down by glutathione beads and subjected to immunoblot analysis . Data are representative of three independent experiments . ( E ) WT or mutant NLRC4 ( CARD , NACHT , or LRR ( leucine - rich repeat ) ) and HA - VDR were expressed in HEK293T cells , immunoprecipitated , and analyzed by immunoblotting with indicated antibodies . * no specific band . Data are representative of three independent experiments . ( F ) WT or mutant VDR ( DBD or LBD ) and Myc - NLRC4 were expressed in HEK293T cells , immunoprecipitated , and analyzed by immunoblotting with indicated antibodies . Data are representative of three independent experiments .

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Figure 5LPS-primed BMDMs were infected with S. Typhimurium at an MOI of 50 for 2h. Lysates were immunoprecipitated (IP) using an anti-VDR antibody and were immunoblotted with the indicated antibodies(A). Whole-cell lysates were shown as the input. Data are representative of three independent experiments.(B-C) Flag-VDR was co-expressed with Myc-NLRC4 in HEK293T cells; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies. Whole-cell lysates were shown as the input. Data are representative of three independent experiments.(D) Purified GST-VDR was incubated with purified His-NLRC4 for 2 h. His-NLRC4- bound to GST-VDR was pulled down by glutathione beads and subjected to immunoblot analysis. Data are representative of three independent experiments.(E) WT or mutant NLRC4 (CARD, NACHT, or LRR (leucine-rich repeat)) and HA-VDR were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies.*no specific band. Data are representative of three independent experiments.(F) WT or mutant VDR (DBD or LBD) and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies. Data are representative of three independent experiments.


Figure 6A - B HEK293T cells were transfected with Flag - NLRC4 , VDR , Myc - FLagellin , and HA - NAIP5 ( A ) or Flag - NLRC4 , VDR , Myc - PrgJ and HA - NAIP2 ( B ) Samples were immunoprecipitated with the anti - Flag and analyzed by immunoblotting with indicated antibodies . HEK293T cells were transfected with Flag - NLRC4 , LBD - VDR , Myc - FLagellin , and HA - NAIP5 ( C ) or Flag - NLRC4 , LBD - VDR , Myc - PrgJ , and HA - NAIP2 ( D ) . Samples were immunoprecipitated with the anti - Flag and analyzed by immunoblotting with indicated antibodies .

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Figure 6A-B HEK293T cells were transfected with Flag-NLRC4, VDR, Myc-FLagellin, and HA-NAIP5 (A) or Flag-NLRC4, VDR, Myc- PrgJ and HA-NAIP2(B) Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.HEK293T cells were transfected with Flag-NLRC4, LBD-VDR, Myc-FLagellin, and HA-NAIP5 (C) or Flag-NLRC4, LBD-VDR, Myc-PrgJ, and HA-NAIP2 (D). Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.


Figure 7B VDR or mutant VDR ( K123A ) and Myc - NLRC4 were expressed in HEK293T cells , immunoprecipitated with the anti - Flag antibody , and analyzed by immunoblotting with indicated antibodies . C Transfected VDR or mutant VDR ( K123A ) and Flag - NLRC4 , Myc - PrgJ , and HA - NAIP2 in HEK293T cells , immunoprecipitated with the anti - HA antibody , and analyzed by immunoblotting with indicated antibodies . D Vdr - / - BMDMs were infected with lentivirus , which expressed Vector ( NC ) , VDR , and VDR - K123A , infected with S . Typhimurium at an MOI of 50 for 2 h , cell lysates ( Lysate ) and culture supernatants ( SN ) were collected and immunoblotted with the indicated antibodies . E - H LDH release ( E ) , IL - 1β secretion ( F ) , IL - 18 secretion ( G ) and TNF - ɑ secretion ( H ) in supernatants . Data are shown as means ± SEM ; determined by Student ' s t - test ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; In each panel , data are representative of at least three independent experiments .

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Figure 7B VDR or mutant VDR(K123A)and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated with the anti-Flag antibody, and analyzed by immunoblotting with indicated antibodies.C Transfected VDR or mutant VDR(K123A) and Flag-NLRC4, Myc- PrgJ, and HA-NAIP2 in HEK293T cells, immunoprecipitated with the anti-HA antibody, and analyzed by immunoblotting with indicated antibodies.D Vdr-/- BMDMs were infected with lentivirus, which expressed Vector(NC), VDR, and VDR-K123A, infected with S. Typhimurium at an MOI of 50 for 2 h, cell lysates(Lysate) and culture supernatants(SN) were collected and immunoblotted with the indicated antibodies.E-H LDH release(E), IL-1β secretion(F), IL-18 secretion (G) and TNF-ɑ secretion (H) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; **, P < 0.01; ***, P <0.001; In each panel, data are representative of at least three independent experiments.


Figure 1A Luciferase complementation imaging ( LCI ) assays showing that BIC1 interacts with BZR1 . nLUC - BZR1 was co - transformed with cLUC - CCA1 , cLUC - TOC1 , cLUC - ELF3 , cLUC - LUX , cLUC - BIC1 , cLUC , respectively , in leaf epidermal cells of Nicotiana benthamiana as indicated . B BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus . Leaf epidermal cells of Nicotiana benthamiana were co - transformed with nYFP - BZR1 and cYFP - BIC1 . BF , bright field . Scale bars represent 20 μm . C In vitro pull - down assays showing BIC1 directly interacts with BZR1 . Purified GST - BIC1 proteins were incubated with MBP or MBP - BZR1 for the MBP pull - down assay . Arrowhead indicates specific bands . D Co - immunoprecipitation ( Co - IP ) assays showing the interaction between BIC1 and BZR1 in vivo . Seedlings were grown for 6 days under long - day conditions . The immunoprecipitates were detected using anti - GFP and anti - MYC antibodies , respectively . * indicate specific bands . The cross - reacting lower band on IP blot is the heavy chain after IP .

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Figure 1A Luciferase complementation imaging (LCI) assays showing that BIC1 interacts with BZR1. nLUC-BZR1 was co-transformed with cLUC-CCA1, cLUC-TOC1, cLUC-ELF3, cLUC-LUX, cLUC-BIC1, cLUC, respectively, in leaf epidermal cells of Nicotiana benthamiana as indicated.B BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-BZR1 and cYFP-BIC1. BF, bright field. Scale bars represent 20 μm.C In vitro pull-down assays showing BIC1 directly interacts with BZR1. Purified GST-BIC1 proteins were incubated with MBP or MBP-BZR1 for the MBP pull-down assay. Arrowhead indicates specific bands.D Co-immunoprecipitation (Co-IP) assays showing the interaction between BIC1 and BZR1 in vivo. Seedlings were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively. * indicate specific bands. The cross-reacting lower band on IP blot is the heavy chain after IP.


Figure 2A , B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment . Col - 0 , 35S : BIC1 - YFP , 35S : BIC1 - Flag and bic1bic2 seedlings were grown for 6 days on 1 / 2 MS medium supplemented with 1 μM brassinazole ( BRZ ) plus different concentrations of epibrassinolide ( eBL ) . Images of the representative seedlings are shown in ( A ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( B ) . Data are means ± SD ; n > 20 . Scale bars , 2 mm . C RT - qPCR analysis of BZR1 - activated gene expression in the bic1bic2 double mutants . The 6 - d - old Col - 0 and bic1bic2 seedlings grown on 1 / 2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL ( + eBL ) or not ( - eBL ) for 1 hour . The PP2A gene served as internal control . Data are means ± SD ( n = 3 ) . D Heat map of BIC1 - regulated genes . The Col - 0 and 35S : BIC1 - YFP seedlings were grown on 1 / 2 MS medium for 10 days . Three biologic replicates were performed . The colored bar beneath the map indicates fold change ( log2 value ) . E BIC1 facilitates the expression of cell elongation genes . RT - qPCR analysis of genes selected in transcriptomic data . The Col - 0 , 35S : BIC1 - YFP and 35S : BIC1 - Flag seedlings were grown for 5 days under long - day conditions . The PP2A gene served as internal control . Data are means ± SD ( n = 3 ) .

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 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 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

Figure 2A, B Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment. Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 seedlings were grown for 6 days on 1/2 MS medium supplemented with 1 μM brassinazole (BRZ) plus different concentrations of epibrassinolide (eBL). Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD; n>20. Scale bars, 2 mm.C RT-qPCR analysis of BZR1-activated gene expression in the bic1bic2 double mutants. The 6-d-old Col-0 and bic1bic2 seedlings grown on 1/2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL (+eBL) or not (-eBL) for 1 hour. The PP2A gene served as internal control. Data are means ± SD (n=3).D Heat map of BIC1-regulated genes. The Col-0 and 35S:BIC1-YFP seedlings were grown on 1/2 MS medium for 10 days. Three biologic replicates were performed. The colored bar beneath the map indicates fold change (log2 value).E BIC1 facilitates the expression of cell elongation genes. RT-qPCR analysis of genes selected in transcriptomic data. The Col-0, 35S:BIC1-YFP and 35S:BIC1-Flag seedlings were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3).


Figure 3A , B BIC1 and BZR1 synergistically promote hypocotyl elongation . The Col - 0 , 35S : BZR1 - MYC , 35S : BIC1 - YFP and 35S : BZR1 - MYC / 35S : BIC1 - YFP plants were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( A ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( B ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm . C - E BR signaling is enhanced in the 35S : BZR1 - MYC / 35S : BIC1 - YFP plants . Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole ( BRZ ) plus a gradient of concentrations of epibrassinolide ( eBL ) under long - day conditions . Images of the representative seedlings when grown with 500 nM eBL ( + eBL ) or not ( - eBL ) are shown in ( C ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( D ) and ( E ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm . F , G BZR1 - mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2 . Seedlings were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( F ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( G ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , n . s . indicates no significant difference , ( Student ' s t - test ) . Scale bar , 2 mm . H BZR1 - mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2 . The Col - 0 , 35S : BZR1 - MYC , bic1bic2 , 35S : BZR1 - MYC / bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured . Data are means ± SD ( n > 20 ) .

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

Figure 3A, B BIC1 and BZR1 synergistically promote hypocotyl elongation. The Col-0, 35S:BZR1-MYC, 35S:BIC1-YFP and 35S:BZR1-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.C-E BR signaling is enhanced in the 35S:BZR1-MYC/35S:BIC1-YFP plants. Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole (BRZ) plus a gradient of concentrations of epibrassinolide (eBL) under long-day conditions. Images of the representative seedlings when grown with 500 nM eBL (+eBL) or not (-eBL) are shown in (C), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (D) and (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.F, G BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (F), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (G). Data are means ± SD (n>20). **p < 0.01, n.s. indicates no significant difference, (Student's t-test). Scale bar, 2 mm.H BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. The Col-0, 35S:BZR1-MYC, bic1bic2, 35S:BZR1-MYC/bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured. Data are means ± SD (n>20).


Figure 4A BR enhances the stability of BIC1 proteins . The 5 - d - old 35S : BIC1 - YFP transgenic plants were treated with 100 μM cycloheximide ( CHX ) or co - treated with 100 μM CHX and 1 μM epibrassinolide ( eBL ) . Samples were collected at indicated time points and BIC1 - YFP protein levels were analyzed by western blots using anti - GFP antibody . Actin was used to verify equal protein loadings . Intensity of the bands was measured using Adobe Photoshop CS3 Extended program . Three independent experiments were performed with similar results . C Transactivation analysis of BIC1 using a yeast assays . The GAL4 DNA - binding domain ( BD ) alone was used as the negative control . SD - W , synthetic dextrose medium lacking Trp ; SD - WH , synthetic dextrose medium lacking both Trp and His . E Transient transcriptional activity analysis in Arabidopsis protoplasts illustrating the transcriptional activation activity of BIC1 . The relative luciferase activities were calculated by normalizing the LUC values against REN . Error bars represent SD ( n = 3 ) . * * p < 0 . 01 , as determined by a Student ' s t - test . G Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 synergistically activate PRE5 promoter . The ProPRE5 : LUC reporter was co - transformed with the indicated effector constructs . The LUC / REN ratio represents the ProPRE5 : LUC activity relative to the internal control ( REN driven by the 35S promoter ) . Error bars represent SD ( n = 3 ) . H , I BIC1 - mediated hypocotyl elongation was reduced by the addition of SRDX motif . Seedlings were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( H ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( I ) . # 1 , # 2 , # 3 represent different 35S : BIC1 - SRDX - Flag transgenic lines . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm .

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

Figure 4A BR enhances the stability of BIC1 proteins. The 5-d-old 35S:BIC1-YFP transgenic plants were treated with 100 μM cycloheximide (CHX) or co-treated with 100 μM CHX and 1 μM epibrassinolide (eBL). Samples were collected at indicated time points and BIC1-YFP protein levels were analyzed by western blots using anti-GFP antibody. Actin was used to verify equal protein loadings. Intensity of the bands was measured using Adobe Photoshop CS3 Extended program. Three independent experiments were performed with similar results.C Transactivation analysis of BIC1 using a yeast assays. The GAL4 DNA-binding domain (BD) alone was used as the negative control. SD-W, synthetic dextrose medium lacking Trp; SD-WH, synthetic dextrose medium lacking both Trp and His.E Transient transcriptional activity analysis in Arabidopsis protoplasts illustrating the transcriptional activation activity of BIC1. The relative luciferase activities were calculated by normalizing the LUC values against REN. Error bars represent SD (n = 3). **p < 0.01, as determined by a Student's t-test.G Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 synergistically activate PRE5 promoter. The ProPRE5:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratio represents the ProPRE5:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3).H, I BIC1-mediated hypocotyl elongation was reduced by the addition of SRDX motif. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (H), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (I). #1, #2, #3 represent different 35S:BIC1-SRDX-Flag transgenic lines. Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


Figure 5LCI assays showing that BIC1 interacts with PIF4 . Leaf epidermal cells of Nicotiana benthamiana were co - transformed with nLUC - BIC1 or nLUC and cLUC - PIF4 or cLUC as indicated . B In vitro pull - down assays showing that BIC1 directly interacts with PIF4 . Purified GST - BIC1 proteins were incubated with MBP or MBP - PIF4 for the MBP pull - down assay . Arrowhead indicates specific bands . C Co - IP assays showing the interaction between BIC1 and PIF4 in vivo . The 35S : BIC1 - YFP , 35S : PIF4 - MYC and 35S : PIF4 - MYC / 35S : BIC1 - YFP transgenic plants were grown for 6 days under long - day conditions . The immunoprecipitates were detected using anti - GFP and anti - MYC antibodies , respectively . D , E Phenotypic analyses showing that BIC1 and PIF4 synergistically promote hypocotyl elongation . The Col - 0 , 35S : BIC1 - YFP , 35S : PIF4 - MYC and 35S : PIF4 - MYC / 35S : BIC1 - YFP plants were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( D ) , and the hypocotyl lengths of the indicated genotypes were measured and are shown in ( E ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm . F RT - qPCR analysis showing that BIC1 and PIF4 synergistically activate PIF4 target genes . The seedlings growth conditions were same as ( D ) . The PP2A gene was used as an internal control . Data are means ± SD ( n = 3 ) . G , H Phenotypic analyses showing that PIF4 - induced hypocotyl elongation is partially . dependent on the function of BIC1 and BIC2 . The Col - 0 , bic1bic2 , PIF4 - MYC and PIF4 - MYC / bic1bic2 plants were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( G ) and the hypocotyl lengths of indicated genotypes were measured and are shown in ( H ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm . I RT - qPCR analysis showing that PIF4 - induced expression of target genes is partially dependent the function of BIC1 and BIC2 . The seedlings growth conditions were same as ( G ) . The PP2A gene was analyzed as an internal control . Data are means ± SD ( n = 3 ) . * * p < 0 . 01 , as determined by Student ' s t - test . J , K Phenotypic analyses showing that BIC1 - induced hypocotyl elongation is largely dependent on the function of PIFs . The Col - 0 , pifQ , 35S : BIC1 - Flag , 35S : BIC1 - flag / pifQ # 7 and BIC1 - flag / pifQ # 8 plants were grown for 5 days under long - day conditions . Images of the representative seedlings are shown in ( J ) and the hypocotyl lengths of indicated genotypes were measured and are shown in ( K ) . Data are means ± SD ( n > 20 ) . * * p < 0 . 01 , as determined by Student ' s t - test . Scale bar , 2 mm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O 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 5LCI assays showing that BIC1 interacts with PIF4. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nLUC-BIC1 or nLUC and cLUC-PIF4 or cLUC as indicated.B In vitro pull-down assays showing that BIC1 directly interacts with PIF4. Purified GST-BIC1 proteins were incubated with MBP or MBP-PIF4 for the MBP pull-down assay. Arrowhead indicates specific bands.C Co-IP assays showing the interaction between BIC1 and PIF4 in vivo. The 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP transgenic plants were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively.D, E Phenotypic analyses showing that BIC1 and PIF4 synergistically promote hypocotyl elongation. The Col-0, 35S:BIC1-YFP, 35S:PIF4-MYC and 35S:PIF4-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (D), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (E). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.F RT-qPCR analysis showing that BIC1 and PIF4 synergistically activate PIF4 target genes. The seedlings growth conditions were same as (D). The PP2A gene was used as an internal control. Data are means ± SD (n=3).G,H Phenotypic analyses showing that PIF4-induced hypocotyl elongation is partially. dependent on the function of BIC1 and BIC2. The Col-0, bic1bic2, PIF4-MYC and PIF4-MYC/bic1bic2 plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (G) and the hypocotyl lengths of indicated genotypes were measured and are shown in (H). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.I RT-qPCR analysis showing that PIF4-induced expression of target genes is partially dependent the function of BIC1 and BIC2. The seedlings growth conditions were same as (G). The PP2A gene was analyzed as an internal control. Data are means ± SD (n=3). **p < 0.01, as determined by Student's t-test.J, K Phenotypic analyses showing that BIC1-induced hypocotyl elongation is largely dependent on the function of PIFs. The Col-0, pifQ, 35S:BIC1-Flag, 35S:BIC1-flag/pifQ #7 and BIC1-flag/pifQ #8 plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (J) and the hypocotyl lengths of indicated genotypes were measured and are shown in (K). Data are means ± SD (n>20). **p < 0.01, as determined by Student's t-test. Scale bar, 2 mm.


Figure 6B ChIP - qPCR analysis showing the association of BIC1 with the promoter regions of BZR1 / PIF4 target genes . The 6 - d - old Col - 0 and 35S : BIC1 - Flag seedlings were used for ChIP assays . C ChIP - qPCR analysis showing that the association of BIC1 with the promoter regions of BZR1 / PIF4 target genes is dependent on PIFs . The 6 - d - old 35S : BIC1 - Flag and 35S : BIC1 - Flag / pifQ seedlings were used for ChIP assays . D ChIP - qPCR analysis showing that the enrichment of BZR1 in the promoter regions of its target genes is dependent on BICs . The 10 - d - old 35S : BZR1 - MYC and 35S : BZR1 - MYC / bic1bic2 seedlings were treated with 1 μM epibrassinolide ( eBL ) for 3 h and then collected for ChIP assays . E ChIP - qPCR analysis showing that the enrichment of PIF4 in the promoter regions of its target genes is dependent on BICs . The 6 - d - old 35S : PIF4 - MYC and 35S : PIF4 - MYC / bic1bic2 seedlings were used for ChIP assays .

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 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 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

Figure 6B ChIP-qPCR analysis showing the association of BIC1 with the promoter regions of BZR1/PIF4 target genes. The 6-d-old Col-0 and 35S:BIC1-Flag seedlings were used for ChIP assays.C ChIP-qPCR analysis showing that the association of BIC1 with the promoter regions of BZR1/PIF4 target genes is dependent on PIFs. The 6-d-old 35S:BIC1-Flag and 35S:BIC1-Flag/pifQ seedlings were used for ChIP assays.D ChIP-qPCR analysis showing that the enrichment of BZR1 in the promoter regions of its target genes is dependent on BICs. The 10-d-old 35S:BZR1-MYC and 35S:BZR1-MYC/bic1bic2 seedlings were treated with 1 μM epibrassinolide (eBL) for 3 h and then collected for ChIP assays.E ChIP-qPCR analysis showing that the enrichment of PIF4 in the promoter regions of its target genes is dependent on BICs. The 6-d-old 35S:PIF4-MYC and 35S:PIF4-MYC/bic1bic2 seedlings were used for ChIP assays.


Figure 7A RT - qPCR analysis showing that BIC1 upregulates the transcriptional expression of PIF4 . The Col - 0 , 35S : BIC1 - YFP , 35S : BIC1 - Flag and bic1bic2 plants were grown for 5 days under long - day conditions . The PP2A gene served as internal control . Data are means ± SD ( n = 3 ) . Different letters indicate significant differences ( Fisher ' s LSD , P < 0 . 05 ) . B Transcript levels of BIC1 and PIF4 in transgenic plants containing a chemical - inducible construct pERGW - BIC1 . 8 - d - old pERGW - BIC1 seedlings were treated with 10 μM estradiol for indicated time points before harvest for RNA extraction and RT - qPCR analysis . The PP2A gene served as internal control . Data are means ± SD ( n = 3 ) . C ChIP - qPCR analysis showing the association of BIC1 with the promoter regions of PIF4 . The top panel shows the diagram of the promoter structure of PIF4 . Arrows indicate the primer positions for ChIP - qPCR . G - box , CACGTG . The 6 - d - old Col - 0 and 35S : BIC1 - Flag seedlings were used for ChIP assays . The chromatin of each sample was immunoprecipitated ( IP ) using an anti - Flag or not ( Mock ) . Precipitated DNA was quantified by qPCR , and DNA enrichment is displayed as the ratio between IP and Mock , normalized to that of PP2A as an internal control . Data are means ± SD ( n = 3 ) . * p < 0 . 05 , as determined by Student ' s t - test . D RT - qPCR analysis showing the transcript levels of endogenous PIF4 in the 5 - d - old Col - 0 , 35S - PIF4 and PIF4 - HA seedlings . The PP2A gene served as internal control . Data are means ± SD ( n = 3 ) . E Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 / PIF4 synergistically activate PIF4 promoter . The ProPIF4 : LUC reporter was co - transformed with the indicated effector constructs . The LUC / REN ratios represent the ProPIF4 : LUC activity relative to the internal control ( REN driven by the 35S promoter ) . Error bars represent SD ( n = 3 ) . * p < 0 . 05 , as determined by Student ' s t - test . F LCI assays showing that TaBIC1 interacts with TaBZR1 . Leaf epidermal cells of Nicotiana benthamiana were co - transformed with nLUC - TaBIC1 or nLUC and cLUC - TaBZR1 or cLUC as indicated . G In vitro pull - down assays showing that TaBIC1 directly interacts with TaBZR1 . Purified GST - TaBIC1 proteins were incubated with MBP or MBP - TaBZR1 for the MBP pull - down assay . Arrowhead indicates specific band . H BiFC assays showing the interaction of TaBIC1 and TaBZR1 in nucleus . Leaf epidermal cells of Nicotiana benthamiana were co - transformed with nYFP - TaBIC1 and cYFP - TaBZR1 . The yellow dots represent interaction signals . BF , bright field . Scale bars , 20 μm .

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

Figure 7A RT-qPCR analysis showing that BIC1 upregulates the transcriptional expression of PIF4. The Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 plants were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n=3). Different letters indicate significant differences (Fisher's LSD, P< 0.05).B Transcript levels of BIC1 and PIF4 in transgenic plants containing a chemical-inducible construct pERGW-BIC1. 8-d-old pERGW-BIC1 seedlings were treated with 10 μM estradiol for indicated time points before harvest for RNA extraction and RT-qPCR analysis. The PP2A gene served as internal control. Data are means ± SD (n=3).C ChIP-qPCR analysis showing the association of BIC1 with the promoter regions of PIF4. The top panel shows the diagram of the promoter structure of PIF4. Arrows indicate the primer positions for ChIP-qPCR. G-box, CACGTG. The 6-d-old Col-0 and 35S:BIC1-Flag seedlings were used for ChIP assays. The chromatin of each sample was immunoprecipitated (IP) using an anti-Flag or not (Mock). Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as the ratio between IP and Mock, normalized to that of PP2A as an internal control. Data are means ± SD (n=3). *p < 0.05, as determined by Student's t-test.D RT-qPCR analysis showing the transcript levels of endogenous PIF4 in the 5-d-old Col-0, 35S-PIF4 and PIF4-HA seedlings. The PP2A gene served as internal control. Data are means ± SD (n=3).E Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1/PIF4 synergistically activate PIF4 promoter. The ProPIF4:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratios represent the ProPIF4:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3). *p < 0.05, as determined by Student's t-test.F LCI assays showing that TaBIC1 interacts with TaBZR1. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nLUC-TaBIC1 or nLUC and cLUC-TaBZR1 or cLUC as indicated.G In vitro pull-down assays showing that TaBIC1 directly interacts with TaBZR1. Purified GST-TaBIC1 proteins were incubated with MBP or MBP-TaBZR1 for the MBP pull-down assay. Arrowhead indicates specific band.H BiFC assays showing the interaction of TaBIC1 and TaBZR1 in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-TaBIC1 and cYFP-TaBZR1. The yellow dots represent interaction signals. BF, bright field. Scale bars, 20 μm.


Figure 1 ( A ) Endogenous FAM134B forms oligomers . FAM134B knockout ( KO ) or wild type ( WT ) 293T cells were treated with 1μM of Thapsigargin ( Tg ) for different time and the cell lysates were prepared and analyzed by Western - blot . ( B ) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE . ( C ) In vitro liposome fragmentation assay . After the injection of recombinant proteins ( 100μg / 100μl ) into the chamber ( 500μl ) , the morphological changes of liposomes were monitored by live imaging for 20 min . The images at different time points as indicated are presented . Scale bars , 10 μm . " ns " means no significance , one - way ANOVA , error bars indicate SEM ( n = 3 ) . ( D ) Quantification of the time between protein addition and liposome fragmentation . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n = 3 ) . ( E , F ) Measurement of the intracellular ER - scission activity . U2OS cells transiently expressing EGFP - FAM134B ( WT ) , EGFP - FAM134B ( 84 - 233 ) or EGFP - FAM134B ( Δ84 - 233 ) at same levels , lysosomal degradation of EGFP - FAM134B was blocked by Bafilomycin A1 ( Baf A1 ) or DMSO . GFP - positive puncta were quantified for each cell in ( F ) . For Each group , at least 30 cells were counted . Scale bars , 10 μm . * * * P < 0 . 001 , " ns " means no significance , one - way ANOVA , error bars indicate SEM . ( G , H ) Measurement of the ER - phagy activity . U2OS cells transiently expressing mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( 84 - 233 ) or mCherry - EGFP - FAM134B ( Δ84 - 233 ) at same levels . ER - phagy is indicated by mCherry - positive but EGFP - negative puncta were quantified for each cell in ( H ) . For mCherry - EGFP - FAM134B ( WT ) , 50 cells were counted ( n = 50 ) ; for mCherry - EGFP - FAM134B ( 84 - 233 ) , n = 56 ; for mCherry - EGFP - FAM134B ( Δ84 - 233 ) , n = 66 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( I ) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co - expressing GFP - SEC61B and FAM134B WT - Flag , FAM134B ( 84 - 233 ) - Flag or FAM134B ( Δ84 - 233 ) - Flag .

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

Figure 1(A) Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild type (WT) 293T cells were treated with 1μM of Thapsigargin (Tg) for different time and the cell lysates were prepared and analyzed by Western-blot.(B) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE.(C) In vitro liposome fragmentation assay. After the injection of recombinant proteins (100μg/100μl) into the chamber (500μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. "ns" means no significance, one-way ANOVA, error bars indicate SEM (n=3).(D) Quantification of the time between protein addition and liposome fragmentation. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).(E, F) Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84-233) or EGFP-FAM134B (Δ84-233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For Each group, at least 30 cells were counted. Scale bars, 10 μm. ***P<0.001, "ns" means no significance, one-way ANOVA, error bars indicate SEM.(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (84-233) or mCherry-EGFP-FAM134B (Δ84-233) at same levels. ER-phagy is indicated by mCherry-positive but EGFP-negative puncta were quantified for each cell in (H). For mCherry-EGFP-FAM134B (WT), 50 cells were counted (n=50); for mCherry-EGFP-FAM134B (84-233), n=56; for mCherry-EGFP-FAM134B (Δ84-233), n=66. Scale bars, 10 μm. The scale bars in the magnification boxes are 2μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(I) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B WT-Flag, FAM134B (84-233)-Flag or FAM134B (Δ84-233)-Flag.


Figure 2 ( A ) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151 . ( B ) Comparison of the self - interaction of FAM134B mono - phosphorylation mutants using co - IP . ( C ) Comparison of the self - interaction of FAM134 tri - phosphorylation mutants using co - IP . ( D ) Comparison of membrane scission activity using in vitro liposome fragmentation assay . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n ≧ 3 ) . ( E ) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co - expressing GFP - SEC61B and FAM134B ( WT ) - Flag , FAM134B ( SA ) - Flag or FAM134B ( SD ) - Flag . ( F , G ) Measurement of the intracellular ER - scission activity . FAM134B knockout ( KO ) U2OS cells was engineered to express EGFP - FAM134B ( WT ) , EGFP - FAM134B ( SA ) or EGFP - FAM134B ( SD ) at endogenous levels , lysosomal degradation of EGFP - FAM134B was blocked by Bafilomycin A1 ( Baf A1 ) . GFP - positive puncta were quantified for each cell in ( G ) . For control , 25 cells were counted ( n = 25 ) ; For EGFP - FAM134B ( WT ) , n = 27 ; for EGFP - FAM134B ( SA ) , n = 29 ; for EGFP - FAM134B ( SD ) , n = 25 . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( H , I ) Measurement of the ER - phagy activity . FAM134B knockout ( KO ) U2OS cells was engineered to express mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( S151A ) or mCherry - EGFP - FAM134B ( S151D ) at endogenous levels . Lysosomal mCherry - positive but GFP - negative puncta were quantified for each cell in ( I ) . For mCherry - EGFP - FAM134B ( WT ) , 25 cells were counted ( n = 25 ) ; for mCherry - EGFP - FAM134B ( SA ) , n = 23 ; for mCherry - EGFP - FAM134B ( SD ) , n = 21 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM .

O O B-PANEL_START 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 B-PANEL_START 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 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 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 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

Figure 2(A) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151.(B) Comparison of the self-interaction of FAM134B mono-phosphorylation mutants using co-IP.(C) Comparison of the self-interaction of FAM134 tri-phosphorylation mutants using co-IP.(D) Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).(E) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B (WT)-Flag, FAM134B (SA)-Flag or FAM134B (SD)-Flag.(F, G) Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells was engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA) or EGFP-FAM134B (SD) at endogenous levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n=25); For EGFP-FAM134B (WT), n=27; for EGFP-FAM134B (SA), n=29; for EGFP-FAM134B (SD), n=25. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(H, I) Measurement of the ER-phagy activity. FAM134B knockout (KO) U2OS cells was engineered to express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (S151A) or mCherry-EGFP-FAM134B (S151D) at endogenous levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (I). For mCherry-EGFP-FAM134B (WT), 25 cells were counted (n=25); for mCherry-EGFP-FAM134B (SA), n=23; for mCherry-EGFP-FAM134B (SD), n=21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.


Figure 3 ( A , B ) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry - FAM134B and BAP31 upon Tg treatment . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . The colocalization was analyzed by Pearson ' s correlation coefficient ( PCC ) in ( B ) . For Ctrl , 16 cells were counted ( n = 16 ) ; for Tg treatment , 18 cells were counted ( n = 18 ) . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( C ) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot . Recombinant proteins for FAM134BWT and FAM134BS151A were purified from E . coil were incubated with purified CAMK2B by IP in kinase buffer . FAM134B phosphorylation was analyzed by a specific antibody recognizing phos - Serine151 of human FAM134B . ( D ) FAM134B S151 phosphorylation in cells treated with CAMK2 activator ( 100nM EB1089 for 1h ) or / and inhibitor ( 10μM KN93 for 2h ) . 293T or SKN - SH ( a cell line derived from neuroblastoma ) cells were treated with drugs as indicated , whole cell lysates were analyzed by phospho - FAM134B ( S151 ) antibody . ( E ) FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells . ( F ) In vitro reconstitution of CAMK2B - FAM134B - mediated membrane fragmentation using liposome assay . Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30 ° C for 10min , the resultant protein mixtures were transferred to chamber coated with liposomes . Liposome fragmentation was monitored by live imaging . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n = 3 ) . ( G , H ) Measurement of the ER - phagy activity . U2OS cells transiently expressing mCherry - EGFP - FAM134B ( WT ) were treated with compounds as indicated . Lysosomal mCherry - positive but GFP - negative puncta were quantified for each cell in ( H ) . For vehicle control , 53 cells were counted ( n = 53 ) , for Ionomycin , n = 56 ; for EB1089 , n = 54 ; for KN - 93 , n = 58 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( I ) FAM134B WT or knockout ( KO ) U2OS cells were treated with CAMK2 activator Ionomycin ( Iono ) , EB1089 or inhibitor KN93 for 24 hours , Western - blot was performed to analyze the proteins as indicated . ( J ) CAMK2B WT or knockdown ( KD ) U2OS cells were treated with 1 μM Thapsigargin ( Tg ) to induce ER stress for different time as indicated . Cells were collected and analyzed for proteins as indicated by Western blot . ( K ) GFP - cleavage assay . CAMK2B WT or knockdown ( KD ) U2OS cells were co - transfected with FAM134B - Flag and GFP - SEC61B , 24 hours post transfection , cells were treated with drugs as indicated and analyzed for GFP - cleavage by Western blot . Note : the levels of Flag - FAM134B , CAMK2B , GFP - SEC61B and Tubulin were analyzed by Western blot using different membranes .

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

Figure 3(A, B) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry-FAM134B and BAP31 upon Tg treatment. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. The colocalization was analyzed by Pearson's correlation coefficient (PCC) in (B). For Ctrl, 16 cells were counted(n=16); for Tg treatment, 18 cells were counted (n=18). ***P<0.001, one-way ANOVA, error bars indicate SEM.(C) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot. Recombinant proteins for FAM134BWT and FAM134BS151A were purified from E. coil were incubated with purified CAMK2B by IP in kinase buffer. FAM134B phosphorylation was analyzed by a specific antibody recognizing phos-Serine151 of human FAM134B.(D) FAM134B S151 phosphorylation in cells treated with CAMK2 activator (100nM EB1089 for 1h) or/and inhibitor (10μM KN93 for 2h). 293T or SKN-SH (a cell line derived from neuroblastoma) cells were treated with drugs as indicated, whole cell lysates were analyzed by phospho-FAM134B (S151) antibody.(E) FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells.(F) In vitro reconstitution of CAMK2B-FAM134B-mediated membrane fragmentation using liposome assay. Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30°C for 10min, the resultant protein mixtures were transferred to chamber coated with liposomes. Liposome fragmentation was monitored by live imaging. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT) were treated with compounds as indicated. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (H). For vehicle control, 53 cells were counted (n=53), for Ionomycin, n=56; for EB1089, n=54; for KN-93, n=58. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(I) FAM134B WT or knockout (KO) U2OS cells were treated with CAMK2 activator Ionomycin (Iono), EB1089 or inhibitor KN93 for 24 hours, Western-blot was performed to analyze the proteins as indicated.(J) CAMK2B WT or knockdown (KD) U2OS cells were treated with 1 μM Thapsigargin (Tg) to induce ER stress for different time as indicated. Cells were collected and analyzed for proteins as indicated by Western blot.(K) GFP-cleavage assay. CAMK2B WT or knockdown (KD) U2OS cells were co-transfected with FAM134B-Flag and GFP-SEC61B, 24 hours post transfection, cells were treated with drugs as indicated and analyzed for GFP-cleavage by Western blot. Note: the levels of Flag-FAM134B, CAMK2B, GFP-SEC61B and Tubulin were analyzed by Western blot using different membranes.


Figure 4 ( A ) Comparison of the self - interaction of FAM134B WT with mutants as indicated using co - IP . SA - G216R was constructed by simultaneously mutating S149 , S151 and S153 to alanine ( SA ) on the basis of FAM134B - G216R . ( B ) In vitro liposome fragmentation assay . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n ≧ 3 ) . ( C , D ) Measurement of the intracellular ER - scission activity of FAM134B WT , G216R , S151A - G216R and SA - G216R . GFP - tagged FAM134B proteins were expressed in U2OS cells at same levels . The autolyosomal degradation of ER fragments labelled by GFP - FAM134B were blocked by Baf A1 or DMSO . GFP - positive puncta were quantified for each cell in ( D ) . For all of the groups , at least 30 cells were included for quantification ; Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( E , F ) Measurement of the ER - phagy activity of FAM134BG216R . U2OS cells transiently expressing mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( G216R ) , mCherry - EGFP - FAM134B ( S151A - G216R ) or mCherry - EGFP - FAM134B ( SA - G216R ) at same levels . Lysosomal mCherry - positive but GFP - negative puncta were quantified for each cell in ( F ) . For WT , 50 cells were counted ( n = 50 ) ; for G216R , n = 55 ; for S151A - G216R , n = 51 ; for SA - G216R , n = 52 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . * P < 0 . 05 , * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM .

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Figure 4(A) Comparison of the self-interaction of FAM134B WT with mutants as indicated using co-IP. SA-G216R was constructed by simultaneously mutating S149, S151 and S153 to alanine (SA) on the basis of FAM134B-G216R.(B) In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).(C, D) Measurement of the intracellular ER-scission activity of FAM134B WT, G216R, S151A-G216R and SA-G216R. GFP-tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolyosomal degradation of ER fragments labelled by GFP-FAM134B were blocked by Baf A1 or DMSO. GFP-positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(E, F) Measurement of the ER-phagy activity of FAM134BG216R. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (G216R), mCherry-EGFP-FAM134B (S151A-G216R) or mCherry-EGFP-FAM134B (SA-G216R) at same levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (F). For WT, 50 cells were counted (n=50); for G216R, n=55; for S151A-G216R, n=51; for SA-G216R, n=52. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. *P<0.05, ***P<0.001, one-way ANOVA, error bars indicate SEM.


Figure 5 ( A , A ' ) Specific expression of FAM134B mRNA ( green ) in sensory neurons ( red ) of P4d vGlut2Cre ; R26lsl - tdTommouse . Scale bars , 100μm . The white dotted line indicates the dorsal root ganglia ( DRG ) where sensory neurons are genetically labeled with Tomato . Insets of ( a ) and ( a ' ) are high magnifications of sensory neurons showing the co - localization of FAM134B mRNA and Tomato . Scale bars , 10 μm . Cultured sensory neurons from DRGs of E14 . 5 vGlut2Cre ; R26lsl - tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP - FAM134B WT ( B ) , G216R ( C ) , S151A - G216R ( D ) , SA - G216R ( E ) , G216R - ΔLIR ( F ) respectively . Infected sensory neurons ( GFP + / Tom + ) are indicated with arrowheads . The nuclei of all cells are shown with DAPI staining . Scale bars , 20 μm . Cultured sensory neurons from DRGs of E14 . 5 vGlut2Cre ; R26lsl - tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 ( G ) Infected sensory neurons ( GFP + / Tom + ) are indicated with arrowheads . The nuclei of all cells are shown with DAPI staining . Scale bars , 20 μm . ( H ) Quantification of the viability of sensory neuron after 48h , 72h and 96h of infection . Sensory neuron viability was calculated as the percentage of transfected sensory neuron ( GFP + / Tom + ) in total sensory neurons ( Tom + ) . For each time point , four biological replicates were included for statistical analysis . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA , error bars indicate SEM . " ns " is the abbreviation for " not significant " . For all of the groups , at least 10 fields of the slides were included .

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Figure 5(A,A') Specific expression of FAM134B mRNA (green) in sensory neurons (red) of P4d vGlut2Cre;R26lsl-tdTommouse. Scale bars, 100μm. The white dotted line indicates the dorsal root ganglia (DRG) where sensory neurons are genetically labeled with Tomato. Insets of (a) and (a') are high magnifications of sensory neurons showing the co-localization of FAM134B mRNA and Tomato. Scale bars, 10 μm.Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP-FAM134B WT (B), G216R(C), S151A-G216R(D), SA-G216R(E), G216R-ΔLIR (F) respectively. Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 (G) Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.(H) Quantification of the viability of sensory neuron after 48h, 72h and 96h of infection. Sensory neuron viability was calculated as the percentage of transfected sensory neuron (GFP+/Tom+) in total sensory neurons (Tom+). For each time point, four biological replicates were included for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA, error bars indicate SEM. "ns" is the abbreviation for "not significant". For all of the groups, at least 10 fields of the slides were included.


Figure 1Representative IF staining of Sox2 ( green ) and Ki67 ( red ) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI ( blue ) on a sagittal section . Dotted lines , basement membrane ; yellow arrowheads mark approximate stem cell boundaries . SCs , stem cells ; TACs , transit amplifying cells ; Ant , Anterior ; Post , Posterior . The mRNA expression profiling on specific markers of stem cells ( B ) and transit amplifying cells captured on P7 incisors CLE followed by analysis using real - time RT - PCR ( C ) . qRT - PCR results are in arbitrary values after normalization for GapDH . Statistics was performed on triplicates using one - way ANOVA followed by Bonferroni ' s test . Representative IF double staining of E - Cadherin ( ECad , green , for cell border ) and AcTub ( red , for the cores of primary cilia ) on a sagittal section . Sample is counterstained with DAPI ( blue ) . 3D reconstructions of ciliary cores of the stem cells and transit amplifying cells based on AcTub immunostaining . Quantification of cilia length ( F ) and volume ( G ) based on 3D reconstruction ( E ) and comparison between those in stem cell and transit amplifying cell regions . Numbers of quantified cilia were indicated ( n ) . Statistics was performed using one - way ANOVA : All error bars were defined in Statistical Analysis section : All quantification and real - time RT - PCR results were presented using style of mean and standard deviation . Tamoxifen induced IFT88 deletion strategy ( top panel ) and representative images of Sox2 ( green ) and Ki67 ( red ) immunostaining on CLE regions of the indicated mouse strains ( bottom panels ) . Dotted lines , basement membrane . Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88 . 3D reconstruction of Sox2 - positive regions based on its immunolabeling of the indicated mouse strains ( I ) and their volume comparison between mouse strains as indicated ( J ) . Statistics was performed using Student ' s t - test : * * , p < 0 . 01 . Data are represented as mean and standard deviation . Representative IF double staining of ADP - ribosylation factor - like protein 13B ( Arl13b , red , K ) or histone deacetylase 6 ( Hdac6 , red , L ) with AcTub ( green ) as a marker of the primary cilium core following stem cell to transit amplifying cell transition .

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

Figure 1Representative IF staining of Sox2 (green) and Ki67 (red) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI (blue) on a sagittal section. Dotted lines, basement membrane; yellow arrowheads mark approximate stem cell boundaries. SCs, stem cells; TACs, transit amplifying cells; Ant, Anterior; Post, Posterior.The mRNA expression profiling on specific markers of stem cells (B) and transit amplifying cells captured on P7 incisors CLE followed by analysis using real-time RT-PCR (C). qRT- PCR results are in arbitrary values after normalization for GapDH. Statistics was performed on triplicates using one-way ANOVA followed by Bonferroni's test.Representative IF double staining of E-Cadherin (ECad, green, for cell border) and AcTub (red, for the cores of primary cilia) on a sagittal section. Sample is counterstained with DAPI (blue).3D reconstructions of ciliary cores of the stem cells and transit amplifying cells based on AcTub immunostaining.Quantification of cilia length (F) and volume (G) based on 3D reconstruction (E) and comparison between those in stem cell and transit amplifying cell regions. Numbers of quantified cilia were indicated (n). Statistics was performed using one-way ANOVA: All error bars were defined in Statistical Analysis section: All quantification and real-time RT-PCR results were presented using style of mean and standard deviation.Tamoxifen induced IFT88 deletion strategy (top panel) and representative images of Sox2 (green) and Ki67 (red) immunostaining on CLE regions of the indicated mouse strains (bottom panels). Dotted lines, basement membrane. Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88.3D reconstruction of Sox2-positive regions based on its immunolabeling of the indicated mouse strains (I) and their volume comparison between mouse strains as indicated (J). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.Representative IF double staining of ADP-ribosylation factor-like protein 13B (Arl13b, red, K) or histone deacetylase 6 (Hdac6, red, L) with AcTub (green) as a marker of the primary cilium core following stem cell to transit amplifying cell transition.


Figure 2Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop ( green ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 . Sample is counterstained with DAPI ( blue ) . Dotted lines , basement membrane . SCs , stem cells ; TACs , transit amplifying cells ; Ams , ameloblasts . 3D reconstruction showing the association of Prom1 ( green ) with AcTub - labeled ( red ) primary cilia in stem cell and transit amplifying cell regions . Note that the expression of Prom1 is not limited primary cilium but also to microvilli . A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region . Green channel transparency was set up to 70 % . Representative IF staining of Prom1 using antibodies directed either its extracellular loop ( clone 13A4 , green ) or cytoplasmic C - terminal end ( Biorbyt , Orb129549 , red ) on transit amplifying cell regions of the WT vs . Prom1 KO mice . Samples are counterstained with DAPI ( blue ) . Note the lack of Prom1 labeling in Prom1 KO mice . The mRNA profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 and 3 ) in cultured CLESCs . qRT - PCR results are in arbitrary values after normalization for GapDH . protein profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 and 3 ) in cultured CLESCs . The immunoblotting was performed with anti - Prom1 C - terminal antibody . β - actin was used as loading control ( F ) . Molecular mass markers are indicated . Representative IF staining of Prom1 ( red ) and Sox2 ( green ) in CLESCs in vitro . Sample is counterstained with DAPI ( blue ) . Arrows indicate colocalisation of Prom1 with low - medium level of Sox2 in the cell nuclei . Subcellular fractionation of CLESCs into cytoplasm / membrane ( C + M ) and nuclear ( N ) fractions . Materials were analyzed by immunoblotting for Prom1 ( H ) and Arl13b ( I ) . As controls , membranes were blotted for anti - Lamin A / C ( nuclei markers ) and GapDH ( cytoplasm marker ) . Molecular mass markers are indicated . Note the presence of full - length Prom1 as well as Arl13b in nuclear fraction .

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 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 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 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 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

Figure 2Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop (green) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Sample is counterstained with DAPI (blue). Dotted lines, basement membrane. SCs, stem cells; TACs, transit amplifying cells; Ams, ameloblasts.3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited primary cilium but also to microvilli. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%.Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice.The mRNA profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. qRT- PCR results are in arbitrary values after normalization for GapDH.protein profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. The immunoblotting was performed with anti-Prom1 C-terminal antibody. β-actin was used as loading control (F). Molecular mass markers are indicated.Representative IF staining of Prom1 (red) and Sox2 (green) in CLESCs in vitro. Sample is counterstained with DAPI (blue). Arrows indicate colocalisation of Prom1 with low-medium level of Sox2 in the cell nuclei.Subcellular fractionation of CLESCs into cytoplasm/membrane (C+M) and nuclear (N) fractions. Materials were analyzed by immunoblotting for Prom1 (H) and Arl13b (I). As controls, membranes were blotted for anti-Lamin A/C (nuclei markers) and GapDH (cytoplasm marker). Molecular mass markers are indicated. Note the presence of full-length Prom1 as well as Arl13b in nuclear fraction.


Figure 3Representative images ( A ) and quantitative analysis ( B ) of IF staining and 3D reconstruction on Sox2 - positive region for WT vs . Prom1 KO mice . Ant : Anterior ; Post : Posterior ( A ) . Statistics was performed using Student ' s t - test : * * , p < 0 . 01 . Data are represented as mean and standard deviation . Representative IF double staining of Sox 2 ( green ) and Ki67 ( red ) on the and transit amplifying cell regions of WT vs . Prom1 KO mice on frontal sections . Samples are counterstained with DAPI ( blue ) . Quantification of Ki67 + cell number in Sox2 - positive region based on IF data from WT and Prom1 KO mice ( C ) . Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3 . Dotted line indicates the CLE .

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Figure 3Representative images (A) and quantitative analysis (B) of IF staining and 3D reconstruction on Sox2-positive region for WT vs. Prom1 KO mice. Ant: Anterior; Post: Posterior (A). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.Representative IF double staining of Sox 2 (green) and Ki67 (red) on the and transit amplifying cell regions of WT vs. Prom1 KO mice on frontal sections. Samples are counterstained with DAPI (blue). Quantification of Ki67+ cell number in Sox2-positive region based on IF data from WT and Prom1 KO mice (C).Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3. Dotted line indicates the CLE.


Figure 4Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice . Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage . Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage . Clonogenicity assay for colony number ( B ) and size ( C ) after the first the passage of CLESCs derived from WT vs . Prom1 KO mice in the absence or presence of SHH ( 100ng / ml ) . Cells were seeded in triplicate on 24 - well plates as 5 , 000 cell / per well . Sphenoid analyses . For these assays , epithelial and mesenchymal cells were mixed at ratio of 5 , 000 : 5 , 000 per well in triplicates on 96 - well plates and grown for 3 days on Matrigel . Representative micrographs of entire well under indicated conditions are shown . Quantification of sphere diameter on samples presented in ( D ) .

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Figure 4Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice. Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage. Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage.Clonogenicity assay for colony number (B) and size (C) after the first the passage of CLESCs derived from WT vs. Prom1 KO mice in the absence or presence of SHH (100ng/ml). Cells were seeded in triplicate on 24-well plates as 5,000 cell/per well.Sphenoid analyses. For these assays, epithelial and mesenchymal cells were mixed at ratio of 5,000:5,000 per well in triplicates on 96-well plates and grown for 3 days on Matrigel. Representative micrographs of entire well under indicated conditions are shown. Quantification of sphere diameter on samples presented in (D).


Figure 5Representative image of CLESCs harbouring the Ki67 - Fucci system showing colored cells at different cell cycle phases . Flow cytometry analysis of Ki67 - Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1 . As a result , > 95 % of the events have been included in the analysis . The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1 . 0 software . Representative IF staining of AcTub ( blue ) on Ki67 - Fucci CLESCs without and with the presence of SHH . Note that the cilium could be visualised ( arrows ) solely when SHH is present . Representative IF double staining of either AcTub ( D , red ) or Arl13b ( E , red ) with Sox2 ( green ) or Ki67 ( green ) in CLESCs in the presence of SHH . Note the expression levels of Sox2 and Ki67 distinguished stem cells and transit amplifying cells , respectively .

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

Figure 5Representative image of CLESCs harbouring the Ki67-Fucci system showing colored cells at different cell cycle phases.Flow cytometry analysis of Ki67-Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1. As a result, >95% of the events have been included in the analysis. The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1.0 software.Representative IF staining of AcTub (blue) on Ki67-Fucci CLESCs without and with the presence of SHH. Note that the cilium could be visualised (arrows) solely when SHH is present.Representative IF double staining of either AcTub (D, red) or Arl13b (E, red) with Sox2 (green) or Ki67 (green) in CLESCs in the presence of SHH. Note the expression levels of Sox2 and Ki67 distinguished stem cells and transit amplifying cells, respectively.