Figure 3B , C Cell death ( B ; LDH , n = 8 ) and viability ( C ; MTT , n = 6 ) assays in T98 cells 42 - hrs post - transfection . NT : not transfected ; EV : empty vector ; n = independent experiments ; * p < 0 . 05 ; unpaired t test ; mean ± SEM . Protein blots of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats ; EV : empty vector ; n = 3 transfectionsImmunofluorescence of intranuclear inclusions for polyGln expressed from Alt . polyGln and Alt . Int . polyGln constructs in HEK293T cells ; scale bar : 50μm ; green arrow heads indicate cells positive for Alt . Int . polyGln intranuclear inclusions ; dashed white box in merge panels indicates the region show in the orthogonal ( Ortho ) zoomed images which show cross section and localization of diffuse polyGln staining and nuclear polyGln ( Arg ) inclusions with alpha tubulin as a cytoplasmic marker ; n = 4 experiments with n > 55 cells per experimentK PolyGln ( Arg ) proteins but not pure polyGln proteins colocalize with nucleophosmin in HEK293T cells ; scale bars : 20µm ; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co - localization of polyGln inclusions and nucleophosmin in three dimensions .

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 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

Figure 3B, C Cell death (B; LDH, n=8) and viability (C; MTT, n=6) assays in T98 cells 42-hrs post-transfection. NT: not transfected; EV: empty vector; n=independent experiments; * p < 0.05; unpaired t test; mean ± SEM.Protein blots of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats; EV: empty vector; n=3 transfectionsImmunofluorescence of intranuclear inclusions for polyGln expressed from Alt. polyGln and Alt. Int. polyGln constructs in HEK293T cells; scale bar: 50μm; green arrow heads indicate cells positive for Alt. Int. polyGln intranuclear inclusions; dashed white box in merge panels indicates the region show in the orthogonal (Ortho) zoomed images which show cross section and localization of diffuse polyGln staining and nuclear polyGln(Arg) inclusions with alpha tubulin as a cytoplasmic marker; n=4 experiments with n>55 cells per experimentK PolyGln(Arg) proteins but not pure polyGln proteins colocalize with nucleophosmin in HEK293T cells; scale bars: 20µm; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co-localization of polyGln inclusions and nucleophosmin in three dimensions.


Figure 4A - C Protein blotting ( A ) and quantification ( B , C ) of polySer RAN proteins in HEK293T cells from interrupted ( Int . 95 ; Int . 102 ) or pure ( Pure 96 ; Pure 104 ) CAG repeats . EV : empty vector , n = 3 transfections , * * p < 0 . 01 , * p < 0 . 05 , unpaired t test , mean ± SEM . D , Immunofluorescence of RAN polySer protein aggregates from CGG interrupted and pure CAG repeat tracts in HEK293T cells ; scale bar : 10μm . n > 20 cells per construct for each experiment ( Exp . ) F , Protein blot of polyAla RAN proteins expressed from pure or interrupted constructs ; n = 3 transfectionsI , J Protein blotting ( I ) and quantification ( J ) of phospho eIF2α ( p - eIF2α ) relative to total eIF2α ; n = 4 transfections , * p < 0 . 05 , unpaired t test , mean ± SD .

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 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 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

Figure 4A-C Protein blotting (A) and quantification (B, C) of polySer RAN proteins in HEK293T cells from interrupted (Int.95; Int.102) or pure (Pure 96; Pure 104) CAG repeats. EV: empty vector, n=3 transfections, ** p<0.01, *p<0.05, unpaired t test, mean ± SEM.D, Immunofluorescence of RAN polySer protein aggregates from CGG interrupted and pure CAG repeat tracts in HEK293T cells; scale bar: 10μm. n>20 cells per construct for each experiment (Exp.)F, Protein blot of polyAla RAN proteins expressed from pure or interrupted constructs; n=3 transfectionsI, J Protein blotting (I) and quantification (J) of phospho eIF2α (p-eIF2α) relative to total eIF2α; n=4 transfections, * p<0.05, unpaired t test, mean ± SD.


Figure 1Phenotypes of 4 - d - old Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown at 22 ° C in darkness ( D ) or continuous W , R , FR , and B light . Scale bar = 1 mm . Hypocotyl lengths of Col , two cbf1 mutants and two CBF1 - OE lines grown at 22 ° C in D or continuous W , R , FR , and B light . Error bars represent SD from 20 seedlings . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col . NS , not significant .

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

Figure 1Phenotypes of 4-d-old Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown at 22°C in darkness (D) or continuous W, R, FR, and B light. Scale bar = 1 mm.   Hypocotyl lengths of Col, two cbf1 mutants and two CBF1-OE lines grown at 22°C in D or continuous W, R, FR, and B light. Error bars represent SD from 20 seedlings. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col. NS, not significant.


Figure 2qRT - PCR analysis showing the relative expression of CBF1 in 4 - d - old wild - type ( Col ) seedlings grown at 22 ° C in darkness ( D ) or continuous W , R , FR , and B light . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . GUS staining of 4 - d - old homozygous CBF1p : GUS transgenic seedlings grown at 22 ° C in D or continuous W , R , FR , and B light . Scale bar = 1 mm . qRT - PCR data showing that CBF1 expression is induced by different light regimes . Wild - type ( Col ) seedlings were first grown at 22 ° C in D for 4 d , and then transferred to continuous W , FR , R , or B light for the indicated times ranging from 1 h to 24 h . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . Immunoblots showing CBF1 protein levels in nuclear fractions from 4 - d - old wild - type ( Col ) seedlings grown at 22 ° C in D or continuous W , R , FR , and B light . Anti - H3 was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot . Immunoblots showing CBF1 - myc protein levels in 4 - d - old CBF1 - myc seedlings grown at 22 ° C in D or continuous W , R , FR , and B light . anti - HSP was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 - myc normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot . F , G Immunoblots showing the levels of CBF1 - myc proteins after exposure to white light . CBF1 - myc seedlings were first grown at 22 ° C for 4 d in D , and then transferred to continuous W light for the indicated time periods within 1 h ( F ) or ranging from 1 h to 24 h ( G ) . anti - HSP was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1 - myc normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot . Immunoblots showing the levels of CBF1 - myc proteins after exposure to FR ( H ) , R ( I ) or B ( J ) light . CBF1 - myc seedlings were first grown at 22 ° C for 4 d in D , and then transferred to continuous FR , R or B light for the indicated time periods ranging from 1 h to 24 h . anti - HSP was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1 - myc normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot .

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

Figure 2qRT-PCR analysis showing the relative expression of CBF1 in 4-d-old wild-type (Col) seedlings grown at 22°C in darkness (D) or continuous W, R, FR, and B light. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).GUS staining of 4-d-old homozygous CBF1p:GUS transgenic seedlings grown at 22°C in D or continuous W, R, FR, and B light. Scale bar = 1 mm.qRT-PCR data showing that CBF1 expression is induced by different light regimes. Wild-type (Col) seedlings were first grown at 22°C in D for 4 d, and then transferred to continuous W, FR, R, or B light for the indicated times ranging from 1 h to 24 h. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).Immunoblots showing CBF1 protein levels in nuclear fractions from 4-d-old wild-type (Col) seedlings grown at 22°C in D or continuous W, R, FR, and B light. Anti-H3 was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.Immunoblots showing CBF1-myc protein levels in 4-d-old CBF1-myc seedlings grown at 22°C in D or continuous W, R, FR, and B light. anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.F, G Immunoblots showing the levels of CBF1-myc proteins after exposure to white light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous W light for the indicated time periods within 1 h (F) or ranging from 1 h to 24 h (G). anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.Immunoblots showing the levels of CBF1-myc proteins after exposure to FR (H), R (I) or B (J) light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous FR, R or B light for the indicated time periods ranging from 1 h to 24 h. anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.


Figure 3In vitro pull - down of PHYA - C1 ( A ) and PHYB - C1 ( B ) with CBF1 . The His - tagged PHYA / B - N ( photosensory domain ) , PHYA / B - C1 ( PAS - related domain ) , and PHYA / B - C2 ( histidine kinase - related domain ) proteins pulled down with GST - tagged CBF1 or GST were detected by anti - His antibody . Input , 6 % of the His - tagged purified target proteins used in pull - down assays . LCI assays showing that CBF1 interacted with phyA in living plant cells . Scale bar = 1 cm . D , E Co - IP assays showing that CBF1 interacted with phyA ( D ) and phyB ( E ) in vivo . ( D ) homozygous CBF1 - myc seedlings were first grown at 22 ° C in continuous FR light for 4 d , then the total proteins were extracted and subsequently treated with 5 - min R light , or 5 - min R light followed by 5 - min FR light ( R + FR ) before immunoprecipitation . ( E ) homozygous CBF1 - myc seedlings were first grown at 22 ° C in continuous R light for 4 d , then the total proteins were extracted and subsequently treated with 5 - min FR light , or 5 - min FR light followed by 5 - min R light ( FR + R ) before immunoprecipitation . After light treatments , total proteins were incubated with anti - c - myc Affinity Gel ( Sigma - Aldrich ) . The total and precipitated proteins were examined by immunoblotting using antibodies against myc , phyA , phyB , and RPN6 , respectively . The numbers below anti - phyA and anti - phyB blots indicate the relative band intensities of co - precipitated phyA or phyB normalized to those of precipitated CBF1 - myc , respectively . The ratio of the first clear band was set to 100 for each blot .

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

Figure 3In vitro pull-down of PHYA-C1 (A) and PHYB-C1 (B) with CBF1. The His-tagged PHYA/B-N (photosensory domain), PHYA/B-C1 (PAS-related domain), and PHYA/B-C2 (histidine kinase-related domain) proteins pulled down with GST-tagged CBF1 or GST were detected by anti-His antibody. Input, 6% of the His-tagged purified target proteins used in pull-down assays.LCI assays showing that CBF1 interacted with phyA in living plant cells. Scale bar = 1 cm.D, E Co-IP assays showing that CBF1 interacted with phyA (D) and phyB (E) in vivo. (D) homozygous CBF1-myc seedlings were first grown at 22°C in continuous FR light for 4 d, then the total proteins were extracted and subsequently treated with 5-min R light, or 5-min R light followed by 5-min FR light (R+FR) before immunoprecipitation. (E) homozygous CBF1-myc seedlings were first grown at 22°C in continuous R light for 4 d, then the total proteins were extracted and subsequently treated with 5-min FR light, or 5-min FR light followed by 5-min R light (FR+R) before immunoprecipitation. After light treatments, total proteins were incubated with anti-c-myc Affinity Gel (Sigma-Aldrich). The total and precipitated proteins were examined by immunoblotting using antibodies against myc, phyA, phyB, and RPN6, respectively. The numbers below anti-phyA and anti-phyB blots indicate the relative band intensities of co-precipitated phyA or phyB normalized to those of precipitated CBF1-myc, respectively. The ratio of the first clear band was set to 100 for each blot.


Figure 4Immunoblots showing the levels of PIF4 and PIF5 proteins at 22 ° C in 4 - d - old Col , cbf1 - 1 , cbf1 - 2 , and CBF1 - myc seedlings grown in darkness ( D ) or continuous W or R light . Anti - RPN6 was used as a sample loading control . numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of RPN6 , respectively . Phenotypes and hypocotyl lengths of 4 - d - old Col , cbf1 - 1 , pif4 pif5 , cbf1 pif4 pif5 , 35S : PIF4 and 35S : PIF4 cbf1 seedlings grown at 22 ° C in continuous W or R light . Error bars represent SD from 20 seedlings . * * P < 0 . 01 and * * * P < 0 . 001 ( two - tailed t - test ) for the indicated pairs of seedlings . Scale bar = 1 mm . NS , not significant . Immunoblots showing the PIF4 protein levels in 4 - d - old Col , 35S : PIF4 , and 35S : PIF4 cbf1 seedlings grown at 22 ° C in D or continuous W or R light . Anti - RPN6 was used as a sample loading control . numbers below the immunoblots indicate the relative band intensities of PIF4 normalized to those of RPN6 , respectively .

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 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 4Immunoblots showing the levels of PIF4 and PIF5 proteins at 22°C in 4-d-old Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown in darkness (D) or continuous W or R light. Anti-RPN6 was used as a sample loading control. numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of RPN6, respectively.Phenotypes and hypocotyl lengths of 4-d-old Col, cbf1-1, pif4 pif5, cbf1 pif4 pif5, 35S:PIF4 and 35S:PIF4 cbf1 seedlings grown at 22°C in continuous W or R light. Error bars represent SD from 20 seedlings. **P < 0.01 and ***P < 0.001 (two-tailed t-test) for the indicated pairs of seedlings. Scale bar = 1 mm. NS, not significant.Immunoblots showing the PIF4 protein levels in 4-d-old Col, 35S:PIF4, and 35S:PIF4 cbf1 seedlings grown at 22°C in D or continuous W or R light. Anti-RPN6 was used as a sample loading control. numbers below the immunoblots indicate the relative band intensities of PIF4 normalized to those of RPN6, respectively.


Figure 5Yeast three - hybrid assays showing that CBF1 represses the interaction of the Pfr - phyB with PIF4 in yeast cells . AD - PIF4 , phyB - BD and CBF1 were expressed in the yeast strain Y190 as indicated . The β - galactosidase activities were measured by liquid culture assays using ONPG as the substrate . Error bars represent SD of three independent yeast clones . Yeast three - hybrid assays showing that CBF1 represses the interaction of the Pfr - phyB with PIF5 ( B ) in yeast cells . AD - PIF4 , phyB - BD and CBF1 were expressed in the yeast strain Y190 as indicated . The β - galactosidase activities were measured by liquid culture assays using ONPG as the substrate . Error bars represent SD of three independent yeast clones . LCI assays showing that CBF1 inhibits phyB interaction with PIF4 ( C ) in plant cells . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . LCI assays showing that CBF1 inhibits phyB interaction with PIF5 ( D ) in plant cells . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . Semi - in vivo pull - down assays showing that CBF1 inhibits phyB interaction with PIF4 ( E ) The total proteins extracted from 4 - d - old 35S : phyB - GFP seedlings grown at 22 ° C in continuous R light served as the bait , and GST - PIF4 fusion proteins served as the prey . Equivalent amounts of phyB - GFP protein extract and GST - PIF4 fusion proteins , and increasing amounts of His - CBF1 were added as indicated before anti - GFP IP assays were performed . The pulled - down proteins were subjected to immunoblotting with antibodies against phyB , GST and His , respectively . Numbers below the anti - GST blots indicate the relative band intensities of co - precipitated GST - PIF4 normalized to those of precipitated phyB - GFP , respectively . The ratio of the first clear band was set to 100 for each blot . Semi - in vivo pull - down assays showing that CBF1 inhibits phyB interaction with PIF5 ( F ) . The total proteins extracted from 4 - d - old 35S : phyB - GFP seedlings grown at 22 ° C in continuous R light served as the bait , and GST - PIF5 fusion proteins served as the prey . Equivalent amounts of phyB - GFP protein extract and GST - PIF5 fusion proteins , and increasing amounts of His - CBF1 were added as indicated before anti - GFP IP assays were performed . The pulled - down proteins were subjected to immunoblotting with antibodies against phyB , GST and His , respectively . Numbers below the anti - GST blots indicate the relative band intensities of co - precipitated GST - PIF5 normalized to those of precipitated phyB - GFP , respectively . The ratio of the first clear band was set to 100 for each blot .

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 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 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 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

Figure 5Yeast three-hybrid assays showing that CBF1 represses the interaction of the Pfr-phyB with PIF4 in yeast cells. AD-PIF4, phyB-BD and CBF1 were expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent SD of three independent yeast clones.Yeast three-hybrid assays showing that CBF1 represses the interaction of the Pfr-phyB with PIF5 (B) in yeast cells. AD-PIF4, phyB-BD and CBF1 were expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent SD of three independent yeast clones.LCI assays showing that CBF1 inhibits phyB interaction with PIF4 (C) in plant cells. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).LCI assays showing that CBF1 inhibits phyB interaction with PIF5 (D) in plant cells. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).Semi-in vivo pull-down assays showing that CBF1 inhibits phyB interaction with PIF4 (E) The total proteins extracted from 4-d-old 35S:phyB-GFP seedlings grown at 22°C in continuous R light served as the bait, and GST-PIF4 fusion proteins served as the prey. Equivalent amounts of phyB-GFP protein extract and GST-PIF4 fusion proteins, and increasing amounts of His-CBF1 were added as indicated before anti-GFP IP assays were performed. The pulled-down proteins were subjected to immunoblotting with antibodies against phyB, GST and His, respectively. Numbers below the anti-GST blots indicate the relative band intensities of co-precipitated GST-PIF4 normalized to those of precipitated phyB-GFP, respectively. The ratio of the first clear band was set to 100 for each blot.Semi-in vivo pull-down assays showing that CBF1 inhibits phyB interaction with PIF5 (F). The total proteins extracted from 4-d-old 35S:phyB-GFP seedlings grown at 22°C in continuous R light served as the bait, and GST-PIF5 fusion proteins served as the prey. Equivalent amounts of phyB-GFP protein extract and GST-PIF5 fusion proteins, and increasing amounts of His-CBF1 were added as indicated before anti-GFP IP assays were performed. The pulled-down proteins were subjected to immunoblotting with antibodies against phyB, GST and His, respectively. Numbers below the anti-GST blots indicate the relative band intensities of co-precipitated GST-PIF5 normalized to those of precipitated phyB-GFP, respectively. The ratio of the first clear band was set to 100 for each blot.


Figure 6Phenotypes of Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown under LD conditions at 22 ° C , 17 ° C and 4 ° C , respectively . The seeds of different genotypes were germinated under LD ( 16 - h - light / 8 - h - dark photoperiod ) conditions at 22 ° C for 2 d , respectively , and then grown under LD conditions at 22 ° C , 17 ° C , or 4 ° C , for additional 6 d . Scale bar = 1 mm . Hypocotyl lengths of Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown under LD conditions at 22 ° C , 17 ° C or 4 ° C . Error bars represent SD from 10 seedlings . * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col . NS , not significant . The ratios of hypocotyl lengths at 17 ° C versus 22 ° C for the indicated seedlings grown under LD conditions . Error bars represent SD from 10 seedlings . * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col . qRT - PCR assays showing the CBF1 transcript levels in Col seedlings grown under LD conditions at 22 ° C , 17 ° C and 4 ° C . Col seeds were germinated under LD conditions at 22 ° C for 2 d , and then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . The samples were collected at ZT8 , and then subjected to qRT - PCR assays . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . E , F Immunoblots showing the protein levels of PIF4 ( E ) and PIF5 ( F ) in Col , cbf1 - 1 , cbf1 - 2 , and CBF1 - myc seedlings grown under LD conditions at 22 ° C , 17 ° C and 4 ° C . The seeds of different genotypes were germinated under LD conditions at 22 ° C for 2 d , and then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . The samples were collected at ZT20 , and then subjected to immunoblotting . Anti - RPN6 was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of loading control , respectively . The ratio of the first band was set to 100 for each blot .

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Figure 6Phenotypes of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C and 4°C, respectively. The seeds of different genotypes were germinated under LD (16-h-light/8-h-dark photoperiod) conditions at 22°C for 2 d, respectively, and then grown under LD conditions at 22°C, 17°C, or 4°C, for additional 6 d. Scale bar = 1 mm.Hypocotyl lengths of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C or 4°C. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col. NS, not significant.The ratios of hypocotyl lengths at 17°C versus 22°C for the indicated seedlings grown under LD conditions. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col.qRT-PCR assays showing the CBF1 transcript levels in Col seedlings grown under LD conditions at 22°C, 17°C and 4°C. Col seeds were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT8, and then subjected to qRT-PCR assays. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).E, F Immunoblots showing the protein levels of PIF4 (E) and PIF5 (F) in Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown under LD conditions at 22°C, 17°C and 4°C. The seeds of different genotypes were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT20, and then subjected to immunoblotting. Anti-RPN6 was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of loading control, respectively. The ratio of the first band was set to 100 for each blot.


Figure 1B Interaction of Atg18 with retromer subunits . Genomically tagged yomCherry - fusions of each retromer subunit were expressed in SEY6210 atg18 ∆ , atg21 ∆ , hsv2 ∆ cells , together with a plasmid expressing Atg18HA3yEGFP . Cell lysates were subjected to immunoprecipitation using RFP - Trap magnetic beads and analyzed by SDS - PAGE and Western blotting against the indicated proteins . D Atg18 - Vps35 interaction . Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26 ∆ strains carrying genomically tagged Atg18HA3yEGFP . Adsorbed proteins were analyzed by SDS - PAGE and Western blotting using the antibodies indicated in brackets . E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled - down Vps35yomCherry . n = 3 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * * P < 0 . 001 . F Atg18 - Vps26 interaction . Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT , SEY6210 vps35 ∆ or SEY6210 vps29 ∆ strains carrying genomically tagged Atg18HA3yEGFP . Adsorbed proteins were analyzed as in D . G Proteins from F were quantified as in E . n = 3 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * * p < 0 . 001 ) .

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Figure 1B Interaction of Atg18 with retromer subunits. Genomically tagged yomCherry-fusions of each retromer subunit were expressed in SEY6210 atg18∆, atg21∆, hsv2∆ cells, together with a plasmid expressing Atg18HA3yEGFP. Cell lysates were subjected to immunoprecipitation using RFP-Trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins.D Atg18-Vps35 interaction. Genomically tagged Vps35yomCherry was pulled down from lysates of SEY6210 WT or SEY6210 vps26∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed by SDS-PAGE and Western blotting using the antibodies indicated in brackets. E Signals from the blots in d were quantified on an infrared fluorescence scanner and normalized to the amount of pulled-down Vps35yomCherry. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** P < 0.001.F Atg18-Vps26 interaction. Genomically tagged Vps26yomCherry was pulled down from lysates of SEY6210 WT, SEY6210 vps35∆ or SEY6210 vps29∆ strains carrying genomically tagged Atg18HA3yEGFP. Adsorbed proteins were analyzed as in D. G Proteins from F were quantified as in E. n=3 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. *** p < 0.001).


Figure 2B Salt - induced vacuole fission . The indicated cells were logarithmically grown in YPD and stained with FM4 - 64 . Vacuole morphology was imaged as in A , before and after a mild salt shock with 0 . 5 M of NaCl for 15 min . The look - up table has been inverted to allow better representation of the clusters of extremely small vacuolar fragments in the SNX mutants . Scale bar : 5 µm . C The number of vacuoles per cell was quantified for the samples from B . n = 3 experiments with at least 100 cells per condition were evaluated ; Error bars represent the SEM .

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Figure 2B Salt-induced vacuole fission. The indicated cells were logarithmically grown in YPD and stained with FM4-64. Vacuole morphology was imaged as in A, before and after a mild salt shock with 0.5 M of NaCl for 15 min. The look-up table has been inverted to allow better representation of the clusters of extremely small vacuolar fragments in the SNX mutants. Scale bar: 5 µm. C The number of vacuoles per cell was quantified for the samples from B. n=3 experiments with at least 100 cells per condition were evaluated; Error bars represent the SEM.


Figure 3A Vps17 labilizes the Atg18 - Vps26 interaction . Wildtype or vps17 ∆ cells expressing ATG18HA3 - yEGFP from a centromeric plasmid were logarithmically grown in YPD . Genomically tagged Vps26yomCherry was pulled down from whole - cell extracts and analyzed for associated Atg18HA3 - yEGFP by SDS - PAGE and Western blotting . Glucose - 6 - phosphate dehydrogenase ( Zwf1 ) serves as a loading control . The intensity of the interacting Atg18HA3 - yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry . Values of the wildtype interaction were used as the reference and set to 1 . n = 4 independent experiments were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM , * * p < 0 . 01 ) B Epistasis of ATG18 and retromer genes concerning vacuolar morphology . The indicated cells were logarithmically grown in YPD medium , stained with FM4 - 64 and calcofluor white C Quantification of vacuole morphology . The number of vacuoles per cell was measured in the cells from B . The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles ( n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student ' s t - test . Bars represent the mean and errors bars the SEM . * * p < 0 .

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Figure 3A Vps17 labilizes the Atg18-Vps26 interaction. Wildtype or vps17∆ cells expressing ATG18HA3-yEGFP from a centromeric plasmid were logarithmically grown in YPD. Genomically tagged Vps26yomCherry was pulled down from whole-cell extracts and analyzed for associated Atg18HA3-yEGFP by SDS-PAGE and Western blotting. Glucose-6-phosphate dehydrogenase (Zwf1) serves as a loading control. The intensity of the interacting Atg18HA3-yEGFP was quantified on a LICOR fluorescence imager and normalized to the amount of Vps26yomCherry. Values of the wildtype interaction were used as the reference and set to 1. n = 4 independent experiments were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM, ** p < 0.01)B Epistasis of ATG18 and retromer genes concerning vacuolar morphology. The indicated cells were logarithmically grown in YPD medium, stained with FM4-64 and calcofluor white C Quantification of vacuole morphology. The number of vacuoles per cell was measured in the cells from B. The graph shows the fractions of cells displaying the indicated numbers of vacuolar vesicles (n = 3 biological experiments with at least 100 cells per condition and experiment were analyzed using an unpaired Student's t-test. Bars represent the mean and errors bars the SEM. ** p < 0.01


Figure 4B Purified Atg18 and CSC - GFP were mixed in a 1 : 1 ratio , incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets .

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Figure 4B Purified Atg18 and CSC-GFP were mixed in a 1:1 ratio, incubated together in PBS and analyzed by native PAGE and Western blotting using the antibodies indicated in brackets.


Figure 5C Pull - down . Cells ( SEY6210 atg18 ∆ , atg21 ∆ ) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3 - yEGFP variants were logarithmically grown in SC - URA media . Vps26yomCherry was pulled down from whole - cell extracts with RFP - trap magnetic beads and analyzed by SDS - PAGE and Western blotting against the indicated proteins . Bands were quantified on a LICOR fluorescence imager . Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry . Vps1 served as a loading control . n = 3 independent replicates . Data were subjected to an unpaired t - test . Bars represent the mean and errors bars the SEM , * * * P < 0 . 001 ) .

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Figure 5C Pull-down. Cells (SEY6210 atg18∆, atg21∆) expressing genomically tagged Vps26yomCherry and the indicated Atg18HA3-yEGFP variants were logarithmically grown in SC-URA media. Vps26yomCherry was pulled down from whole-cell extracts with RFP-trap magnetic beads and analyzed by SDS-PAGE and Western blotting against the indicated proteins. Bands were quantified on a LICOR fluorescence imager. Signals of Atg18HA3yEGFP were normalized relative to those of Vps26yomCherry. Vps1 served as a loading control. n = 3 independent replicates. Data were subjected to an unpaired t-test. Bars represent the mean and errors bars the SEM, *** P < 0.001).


Figure 6A , Effect of Atg18T56E on salt - induced vacuole fission . Cells expressing Atg18WTHA3 - yEGFP or Atg18T56E - HA3 - yEGFP from centromeric plasmids in a SEY6210 atg18 ∆ , atg21 ∆ strain were logarithmically grown in SD - URA medium . They were stained and imaged before and after the induction of vacuole fission by a short salt shock Calcofluor white - stained cell walls are only represented in the merge ( blue ) . Scale bar : 5 µm . B , Quantification of the number of vacuoles per cell from A , n = 3 biological experiments with at least 100 cells per condition were scored ; bars represent the mean and error bars the SEM . C , Epistasis of atg18T56E over a vps17 ∆ mutation . The indicated variants of Atg18 - HA3 - yEGFP were expressed from plasmids in a SEY6210 atg18 ∆ , atg21 ∆ , vps17 ∆ strain . Cells were logarithmically grown in SD - URA and imaged Scale bar : 5 µm . D , Quantification of the experiments from C , n = 3 independent experiments with at least 100 cells per condition scored ; bars represent the mean and error bars the SEM .

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Figure 6A, Effect of Atg18T56E on salt-induced vacuole fission. Cells expressing Atg18WTHA3-yEGFP or Atg18T56E-HA3-yEGFP from centromeric plasmids in a SEY6210 atg18∆, atg21∆ strain were logarithmically grown in SD-URA medium. They were stained and imaged before and after the induction of vacuole fission by a short salt shock Calcofluor white-stained cell walls are only represented in the merge (blue). Scale bar: 5 µm. B, Quantification of the number of vacuoles per cell from A, n = 3 biological experiments with at least 100 cells per condition were scored; bars represent the mean and error bars the SEM. C, Epistasis of atg18T56E over a vps17∆ mutation. The indicated variants of Atg18-HA3-yEGFP were expressed from plasmids in a SEY6210 atg18∆, atg21∆, vps17∆ strain. Cells were logarithmically grown in SD-URA and imaged Scale bar: 5 µm. D, Quantification of the experiments from C, n = 3 independent experiments with at least 100 cells per condition scored; bars represent the mean and error bars the SEM .


Figure 7A . Atg18 - dependent recruitment of CSC - GFP to small liposomes . SUVs were incubated ( 10 min , 25 ° C ) with purified CSC ( 1 . 5 µM ) alone or in combination with the indicated recombinant Atg18 variants ( 1 . 5 µM ) . The vesicles were sedimented by centrifugation and supernatants ( Sup . ) and pellets were analyzed by SDS - PAGE and Coomassie staining . B . Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner . n = 3 independent experiments . Bars represent the mean and error bars the SEM , * * p < 0 . 01 ; * p < 0 . 05 . C . Quantification as in B , but for the Atg18 variants .

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Figure 7A. Atg18-dependent recruitment of CSC-GFP to small liposomes. SUVs were incubated (10 min, 25°C) with purified CSC (1.5 µM) alone or in combination with the indicated recombinant Atg18 variants (1.5 µM). The vesicles were sedimented by centrifugation and supernatants (Sup.) and pellets were analyzed by SDS-PAGE and Coomassie staining. B. Quantification of the Coomassie signals for Vps35 by densitometry on a LICOR Odyssey scanner. n = 3 independent experiments. Bars represent the mean and error bars the SEM, ** p < 0.01; * p < 0.05. C. Quantification as in B, but for the Atg18 variants.


Figure 9A Colocalization of WIPI1 with Vps26A . The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells , from which endogenous WIPI1 had been deleted ( WIPI1 - KO ) . The cells were analyzed by confocal microscopy . Scale bars : 10 μm . Insets show enlargements of the outlined areas . B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders ' colocalization coefficient M2 . The red line indicates the mean ; n = 120 cells per condition , pooled from three independent experiments . P - values were calculated by t - test ( analysis performed with 99 % confidence * * * p < 0 . 0001 ) . C Expression levels of WIPI1mCherry variants . Lysates ( 50 μg of protein per sample ) from the cells in A were analyzed by SDS - PAGE and Western blot against WIPI1 and tubulin . D Depletion of VPS35 . HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 ( VPS35 KD ) or a control siRNA pool . Lysates ( 50 μg per sample ) from the cells were analyzed by SDS - PAGE and Western blot against Vps35 and tubulin . E Blots from D were quantified on a LICOR Odyssey fluorescence imager . n = 3 . Red lines indicate the means , n = 3 independent experiments , using a Welch ' s t - test statistical analysis . Bars represent the mean and error bars the SD . F Cells from D were analyzed by confocal microscopy 18 h after transfection . Scale bar : 10 μm . G Quantification of tubule length in the cells from F . Data are means ± SD . of n = 210 tubules per condition , from three independent experiments . p values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

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

Figure 9A Colocalization of WIPI1 with Vps26A. The indicated WIPI1mCherry variants and VPS26EGFP were expressed for 18 h in HK2 cells, from which endogenous WIPI1 had been deleted (WIPI1-KO). The cells were analyzed by confocal microscopy. Scale bars: 10 μm. Insets show enlargements of the outlined areas. B Colocalization between WIPI1 variants and Vps26 was quantified in the cells from A using the Manders' colocalization coefficient M2. The red line indicates the mean; n = 120 cells per condition, pooled from three independent experiments. P-values were calculated by t-test (analysis performed with 99% confidence ***p < 0.0001). C Expression levels of WIPI1mCherry variants. Lysates (50 μg of protein per sample) from the cells in A were analyzed by SDS-PAGE and Western blot against WIPI1 and tubulin.D Depletion of VPS35. HK2 cells expressing the indicated WIPI1eGFP variants were transfected with siRNA against VPS35 (VPS35 KD) or a control siRNA pool. Lysates (50 μg per sample) from the cells were analyzed by SDS-PAGE and Western blot against Vps35 and tubulin. E Blots from D were quantified on a LICOR Odyssey fluorescence imager. n=3. Red lines indicate the means, n = 3 independent experiments, using a Welch's t-test statistical analysis. Bars represent the mean and error bars the SD.F Cells from D were analyzed by confocal microscopy 18 h after transfection. Scale bar: 10 μm. G Quantification of tubule length in the cells from F. Data are means ± SD. of n=210 tubules per condition, from three independent experiments. p values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.


Figure 10A , Tf recycling . WIPI1 - KO cells were transfected with WIPI1WT - eGFP , WIPI1S69E - eGFP or WIPI1S69A - eGFP for 18h . Then , they were serum - starved for 1 h , loaded with Alexa Fluor 568 - conjugated Tf , chased at 37 ° C for 1 h without labeled Tf , and analyzed by confocal microscopy . Scale bar : 10 μm . White dashed lines delineate the circumference of the cells . B , Quantification of Tf - fluorescence in the cells from a that expressed WIPI1 variants . Total cell fluorescence was integrated and corrected for background fluorescence . Mean values ± SD are shown . n = 3 independent experiments with a total of 150 cells analyzed per condition . P values were calculated by unpaired Student ' s t - test . 99 % confidence : * * * = p < 0 . 0001 .

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 10A, Tf recycling. WIPI1-KO cells were transfected with WIPI1WT-eGFP, WIPI1S69E-eGFP or WIPI1S69A-eGFP for 18h. Then, they were serum-starved for 1 h, loaded with Alexa Fluor 568-conjugated Tf, chased at 37°C for 1 h without labeled Tf, and analyzed by confocal microscopy. Scale bar: 10 μm. White dashed lines delineate the circumference of the cells. B, Quantification of Tf-fluorescence in the cells from a that expressed WIPI1 variants. Total cell fluorescence was integrated and corrected for background fluorescence. Mean values ± SD are shown. n = 3 independent experiments with a total of 150 cells analyzed per condition. P values were calculated by unpaired Student's t-test. 99% confidence: *** = p < 0.0001.


Figure 1B . BJ ESCs stably co - expressing H2B - GFP ( green ) and mCherry - H2B ( red ) , hereafter named BJH2B - 2FPs . C . ChIP - qPCR analysis of GFP enrichment inside different repetitive elements ( ERV , MajSat , IAPs ) used as H3K9me3 domain controls , or at the Hoxa11 gene used as a H3K27me3 domain control , or on transcribed genes ( Gapdh , Pou5f1 , Actb ) or actively transcribed gene promoter ( Pou5f1 - promoter ) in BJ WT and BJH2B - GFP ESCs . Gene names are color - coded in red whether they are associated to heterochromatic domains ( enriched in H3K9me3 ) ; in black to Polycomb domains ( enriched in H3K27me3 ) ; in blue to active chromatin domains ( enriched in H3K4me3 ) . Data are represented as relative enrichments of H2B - GFP versus histone H2B . Data are means ± s . d ( n = 3 biological replicates for GFP and histone H2B ) . E . Left panel , in vivo FLIM - FRET assay in BJH2B - 2FPs stably co - expressing H2B - GFP ( green ) and mCherry - H2B ( red ) . Mean FRET efficiency is displayed using a continuous pseudo - colour scale from 0 to 40 % . Magnification of the FRET map , with discrete high FRET regions indicated by white arrowheads . Scale bars , 5 µm . Right panel , mean distribution of the FRET efficiency related to the pixel fraction from BJH2B - 2FPs cells ( n = 384 cells ) .

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 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

Figure 1B. BJ ESCs stably co-expressing H2B-GFP (green) and mCherry-H2B (red), hereafter named BJH2B-2FPs. C. ChIP-qPCR analysis of GFP enrichment inside different repetitive elements (ERV, MajSat, IAPs) used as H3K9me3 domain controls, or at the Hoxa11 gene used as a H3K27me3 domain control, or on transcribed genes (Gapdh, Pou5f1, Actb) or actively transcribed gene promoter (Pou5f1-promoter) in BJ WT and BJH2B-GFP ESCs. Gene names are color-coded in red whether they are associated to heterochromatic domains (enriched in H3K9me3); in black to Polycomb domains (enriched in H3K27me3); in blue to active chromatin domains (enriched in H3K4me3). Data are represented as relative enrichments of H2B-GFP versus histone H2B. Data are means ± s.d (n=3 biological replicates for GFP and histone H2B).E. Left panel, in vivo FLIM-FRET assay in BJH2B-2FPs stably co-expressing H2B-GFP (green) and mCherry-H2B (red). Mean FRET efficiency is displayed using a continuous pseudo-colour scale from 0 to 40%. Magnification of the FRET map, with discrete high FRET regions indicated by white arrowheads. Scale bars, 5 µm. Right panel, mean distribution of the FRET efficiency related to the pixel fraction from BJH2B-2FPs cells (n = 384 cells).


Figure 2A . Top and bottom panels depict a representative nucleus of BJH2B - 2FPs ESC by GFP intensity ( nucleosome concentration ) and FRET efficiency , respectively . Yellow arrowheads : regions with high nucleosome density but low FRET efficiency ; Orange arrowheads : regions with low nucleosome density but high FRET efficiency . Scale bars , 10 μm . The right panel correlates FRET efficiency with GFP fluorescence intensity ( Pearson correlation coefficient , r = - 0 . 07 , n = 106 cells ) . F . Top and bottom panels depict a representative nucleus of BJH2B - 2FPs ESC after 15 min of ATP depletion by GFP intensity ( i . e . , nucleosome concentration ) and FRET efficiency , respectively . Yellow arrows indicate nucleosome - rich foci associated with high levels of FRET . Scale bars , 10 µm . The FRET efficiencies ( % ) from nucleosome - rich foci in untreated and ATP - depleted cells are depicted as Box - and - Whisker plots . The thick line represents median , the boxes correspond to the FRET efficiency ( % ) values from the 25 - 75th percentiles of the median , and the whiskers cover the Minimum to Maximum value range . * * * * , p < 0 . 0001 ( Mann - Whitney test , n = 61 cells ) . Mean distribution of the FRET efficiency of BJH2B - 2FPs from nucleosome - rich foci in untreated ( black , n = 162 ) and ATP - depleted cells ( red , n = 61 cells ) . * * * * , p = 2 . 2e - 16 ; K - S test . G . Left panel , representative image of in vivo FLIM - FRET measurements from differentiated 3T3 cells co - expressing H2B - GFP and mCherry - H2B . Yellow arrows indicate high FRET efficiencies at H2B - GFP bright chromocenters . Scale bars , 10 µm . Right panel , comparison of the FRET efficiency ( % ) from ESCs ( blue ) and 3T3 cells ( white ) in the GFP - brightest foci ( nucleosome - rich foci ) . FRET efficiency ( % ) was represented as Box - and - Whisker plots . The thick line represents median , the boxes correspond to the FRET % values from the 25 - 75th percentiles of the median , and the whiskers cover the Minimum to Maximum value range . * * * * , p < 0 . 0001 ( Mann - Whitney test , n = 42 cells ) .

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

Figure 2A. Top and bottom panels depict a representative nucleus of BJH2B-2FPs ESC by GFP intensity (nucleosome concentration) and FRET efficiency, respectively. Yellow arrowheads: regions with high nucleosome density but low FRET efficiency; Orange arrowheads: regions with low nucleosome density but high FRET efficiency. Scale bars,10 μm. The right panel correlates FRET efficiency with GFP fluorescence intensity (Pearson correlation coefficient, r = -0.07, n = 106 cells).F. Top and bottom panels depict a representative nucleus of BJH2B-2FPs ESC after 15 min of ATP depletion by GFP intensity (i.e., nucleosome concentration) and FRET efficiency, respectively. Yellow arrows indicate nucleosome-rich foci associated with high levels of FRET. Scale bars,10 µm. The FRET efficiencies (%) from nucleosome-rich foci in untreated and ATP-depleted cells are depicted as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET efficiency (%) values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range. ****, p < 0.0001 (Mann-Whitney test, n = 61 cells). Mean distribution of the FRET efficiency of BJH2B-2FPs from nucleosome-rich foci in untreated (black, n = 162) and ATP-depleted cells (red, n = 61 cells). ****, p = 2.2e-16; K-S test.G. Left panel, representative image of in vivo FLIM-FRET measurements from differentiated 3T3 cells co-expressing H2B-GFP and mCherry-H2B. Yellow arrows indicate high FRET efficiencies at H2B-GFP bright chromocenters. Scale bars, 10 µm. Right panel, comparison of the FRET efficiency (%) from ESCs (blue) and 3T3 cells (white) in the GFP-brightest foci (nucleosome-rich foci). FRET efficiency (%) was represented as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range. ****, p < 0.0001 (Mann-Whitney test, n = 42 cells).


Figure 3A . Expression of mTagBFP - tagged HP1α in living BJH2B - 2FPs and segmentation of HP1α positive foci . Yellow arrowheads : chromocenters enriched in H2B - GFP ( left panel ) , in mTagBFP - HP1α ( middle panel ) , and following segmentation ( right panel ) . B . Box plot of the mean FRET efficiencies of all nucleosome - rich foci ( black , n = 68 cells ) and foci enriched in HP1α ( blue , n = 68 cells ) . FRET efficiency ( % ) was represented as Box - and - Whisker plots . The thick line represents median , the boxes correspond to the FRET % values from the 25 - 75th percentiles of the median , and the whiskers cover the 10 - 90 percentiles value range . * * * * p < 0 . 0001 , Mann - Whitney test . D . Representative image of the FRET efficiency map of interphase and mitotic ESCs ( top and bottom panels , respectively ) after fixation with 4 % PFA . The mean FRET efficiency is displayed using a continuous pseudo - colour scale from 0 to 80 % . E . Mean distribution of FRET efficiency from nucleosome - rich foci in living cells ( green , n = 162 cells ) , and fixed cells ( black , n = 157 cells ) . * * * * , p = 2 . 2e - 16 ; K - S test . Right panel , box plot of the mean FRET efficiency from nucleosome - rich foci in living and fixed ESCs . FRET efficiency ( % ) was represented as Box - and - Whisker plots . The thick line represents median , the boxes correspond to the FRET % values from the 25 - 75th percentiles of the median , and the whiskers cover the Minimum to Maximum value range . * * * * p < 0 . 0001 , Mann - Whitney 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 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 O 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. Expression of mTagBFP-tagged HP1α in living BJH2B-2FPs and segmentation of HP1α positive foci. Yellow arrowheads: chromocenters enriched in H2B-GFP (left panel), in mTagBFP-HP1α (middle panel), and following segmentation (right panel). B. Box plot of the mean FRET efficiencies of all nucleosome-rich foci (black, n = 68 cells) and foci enriched in HP1α (blue, n = 68 cells). FRET efficiency (%) was represented as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the 10-90 percentiles value range. **** p < 0.0001, Mann-Whitney test.D. Representative image of the FRET efficiency map of interphase and mitotic ESCs (top and bottom panels, respectively) after fixation with 4% PFA. The mean FRET efficiency is displayed using a continuous pseudo-colour scale from 0 to 80%.E. Mean distribution of FRET efficiency from nucleosome-rich foci in living cells (green, n = 162 cells), and fixed cells (black, n = 157 cells). ****, p = 2.2e-16; K-S test. Right panel, box plot of the mean FRET efficiency from nucleosome-rich foci in living and fixed ESCs. FRET efficiency (%) was represented as Box-and-Whisker plots. The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range. **** p < 0.0001, Mann-Whitney test.


Figure 4A . Total cell extracts from BJH2B - 2FPs cells incubated during 24h with untargeted siRNA or an siRNA targeting HP1α , analyzed by western blotting with an antiserum against HP1α . Loading control was performed by red ponceau staining . C . Left panel , mean distribution of the FRET efficiency from nucleosome - rich foci of BJH2B - 2FPs cells incubated with a control siRNA ( black , n = 101 cells ) or an siRNA against HP1β ( green , n = 136 cells ) ; * , p : 0 . 02651 ; K - S test . Right panel , box plot of the mean FRET efficiency from nucleosome - rich foci of living of BJH2B - 2FPs cells incubated with a control siRNA ( grey , n = 101 cells ) or an siRNA against HP1β ( green , n = 136 cells ) . Box - and - Whisker plots represent the FRET efficiency ( % ) . The thick line represents median , the boxes correspond to the FRET % values from the 25 - 75th percentiles of the median , and the whiskers cover the Minimum to Maximum value range . E . Left panel , representative images of immunostaining for H4K20me3 in control ESCs ( top row ) and ESCs treated with A - 196 during 2 days ( bottom row ) . Scale bars , 10 µm . Right panel , Box - and - Whisker plots represent the normalized fluorescence intensity of immunostaining for H4K20me3 . The box plots indicate median values ( middle lines ) , mean values ( middle crosses ) , first and third quartiles ( box edges ) and the whiskers cover the minimum to maximum value range of the fluorescence intensity . Data are means of n = 2 biological replicates . Number of nuclei = 76 / 60 for control ESCs / A - 196 treated ESCs . F . Total cell extracts from untreated , DMSO treated or A - 196 treated BJH2B - 2FPs during 24h or 48h , analysed by western blotting with antibody against H4K20me3 . Arrowhead indicates the H4K20me3 - specific bands . The asterisk indicates unspecific bands .

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

Figure 4A. Total cell extracts from BJH2B-2FPs cells incubated during 24h with untargeted siRNA or an siRNA targeting HP1α, analyzed by western blotting with an antiserum against HP1α. Loading control was performed by red ponceau staining.C. Left panel, mean distribution of the FRET efficiency from nucleosome-rich foci of BJH2B-2FPs cells incubated with a control siRNA (black, n = 101 cells) or an siRNA against HP1β (green, n = 136 cells); *, p: 0.02651; K-S test. Right panel, box plot of the mean FRET efficiency from nucleosome-rich foci of living of BJH2B-2FPs cells incubated with a control siRNA (grey, n = 101 cells) or an siRNA against HP1β (green, n = 136 cells). Box-and-Whisker plots represent the FRET efficiency (%). The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the Minimum to Maximum value range.E. Left panel, representative images of immunostaining for H4K20me3 in control ESCs (top row) and ESCs treated with A-196 during 2 days (bottom row). Scale bars, 10 µm. Right panel, Box-and-Whisker plots represent the normalized fluorescence intensity of immunostaining for H4K20me3. The box plots indicate median values (middle lines), mean values (middle crosses), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the fluorescence intensity. Data are means of n=2 biological replicates. Number of nuclei = 76/60 for control ESCs/A-196 treated ESCs.F. Total cell extracts from untreated, DMSO treated or A-196 treated BJH2B-2FPs during 24h or 48h, analysed by western blotting with antibody against H4K20me3. Arrowhead indicates the H4K20me3-specific bands. The asterisk indicates unspecific bands.


Figure 5A . Representative images of immunostainings for Ki - 67 in mitotic WT , control ( CTRL ) and Mki67 - / - ESCs . Scale bar , 10 µm . B . Box - and - Whisker plots represent the FRET efficiency ( % ) from control mitotic ESCs ( n = 12 cells ) and from three different Mki67 - / - ESCs clones named # 1 ( n = 10 cells ) , # 2 ( n = 9 cells ) and # 3 ( n = 8 cells ) . The thick line represents median , the boxes correspond to the FRET % values from the 25 - 75th percentiles of the median , and the whiskers cover the 10 - 90 percentiles value range ; * * * , p = 0 . 0007 ; * * * * , p < 0 . 0001 , Mann - Whitney test . C . Left panel , in vivo FLIM - FRET measurements in interphase BJH2B - 2FPs control ESCs ( top ) and BJH2B - 2FPs Mki67 - / - ESCs ( clone # 1 ) . Mean FRET efficiency is displayed using a continuous pseudo - colour scale from 0 to 25 % . Scale bars , 10 µm . Right panel , box - and - Whisker plots represent the FRET efficiency ( % ) in WT ESCs ( n = 77 cells ) and Mki67 - / - ESCs clones clone # 1 ( n = 86 cells ) , # 2 ( n = 95 cells ) and # 3 ( n = 110 cells ) . The thick line represents median , the boxes correspond to the FRET % values from the 25 - 75th percentiles of the median , and the whiskers cover the 10 - 90 percentiles value range . * * * * , p < 0 . 0001 , Mann - Whitney test .

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Figure 5A. Representative images of immunostainings for Ki-67 in mitotic WT, control (CTRL) and Mki67-/- ESCs. Scale bar, 10 µm.B. Box-and-Whisker plots represent the FRET efficiency (%) from control mitotic ESCs (n = 12 cells) and from three different Mki67-/- ESCs clones named #1 (n = 10 cells), #2 (n = 9 cells) and #3 (n = 8 cells). The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the 10-90 percentiles value range; ***, p = 0.0007; ****, p < 0.0001, Mann-Whitney test.C. Left panel, in vivo FLIM-FRET measurements in interphase BJH2B-2FPs control ESCs (top) and BJH2B-2FPs Mki67-/- ESCs (clone #1). Mean FRET efficiency is displayed using a continuous pseudo-colour scale from 0 to 25%. Scale bars, 10 µm. Right panel, box-and-Whisker plots represent the FRET efficiency (%) in WT ESCs (n = 77 cells) and Mki67-/- ESCs clones clone #1 (n = 86 cells), #2 (n = 95 cells) and #3 (n = 110 cells). The thick line represents median, the boxes correspond to the FRET % values from the 25-75th percentiles of the median, and the whiskers cover the 10-90 percentiles value range. ****, p < 0.0001, Mann-Whitney test.


Figure 6B . Left panels , nucleosome - rich foci segmentation in naive ESCs and EpiLCs using the FRENETIC tool . Nuclei are outlined with yellow dashed lines . Scale bars , 10µm . Right panel , Box - and - Whisker plots of the mean number of foci per nucleus in naive BJH2B - 2FPs ESCs ( n = 150 cells ) and in primed EpiLCs ( n = 137 cells ) . The thick line represents median , the boxes correspond to the number of foci from the 25 - 75th percentiles of the medianC . Left panel , Box - and - Whisker plots represent the FRET efficiencies ( % ) from naive ESCs ( blue , n = 162 cells ) and EpiLCs ( red , n = 266 cells ) . The box plots indicate median values ( middle lines ) , first and third quartiles ( box edges ) and the whiskers cover the minimum to maximum value range of the FRET efficiency ; ns , p = 0 . 29 , Mann - Whitney test . Right panel , mean distribution of the FRET efficiency related to the pixel fraction in nucleosome - rich foci from naive ESCs ( blue , n = 162 cells ) and EpiLCs ( red , n = 266 cells ) . D . Mean distribution of the FRET efficiency related to the pixel fraction in nucleosome - rich foci from EpiLCs at different time points of cellular induction ( black , + 24h n = 57 cells ; orange , + 48h , n = 48 cells ; red , + 72h n = 266 cells ) . * * * * , p = 2 . 2e - 16 ; K - S test . E . Total cell extracts from naive ESCs and EpiLCs ( + 72h ) analyzed by western blot with antiserum against H4K20me3 . Short and long exposures of the signal are shown . Loading control was achieved by Ponceau staining . F . Left panel , mean distribution of the FRET efficiency related to the pixel fraction in nucleosome - rich foci from untreated EpiLCs ( black , n = 124 cells ) and A - 196 EpiLCs treated cells ( red , n = 113 cells ) . * * * * , p = 2 . 2e - 16 ; K - S test . Right panel , Box - and - Whisker plots indicate the FRET efficiency ( % ) from untreated EpiLCs ( black , n = 124 cells ) and treated with A - 196 during 2 days ( red , n = 113 cells ) . The box plots indicate median values ( middle lines ) , first and third quartiles ( box edges ) and the whiskers cover the minimum to maximum value range of the FRET efficiency .

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Figure 6B. Left panels, nucleosome-rich foci segmentation in naive ESCs and EpiLCs using the FRENETIC tool. Nuclei are outlined with yellow dashed lines. Scale bars, 10µm. Right panel, Box-and-Whisker plots of the mean number of foci per nucleus in naive BJH2B-2FPs ESCs (n = 150 cells) and in primed EpiLCs (n = 137 cells). The thick line represents median, the boxes correspond to the number of foci from the 25-75th percentiles of the medianC. Left panel, Box-and-Whisker plots represent the FRET efficiencies (%) from naive ESCs (blue, n = 162 cells) and EpiLCs (red, n = 266 cells). The box plots indicate median values (middle lines), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the FRET efficiency; ns, p = 0.29, Mann-Whitney test. Right panel, mean distribution of the FRET efficiency related to the pixel fraction in nucleosome-rich foci from naive ESCs (blue, n = 162 cells) and EpiLCs (red, n = 266 cells).D. Mean distribution of the FRET efficiency related to the pixel fraction in nucleosome-rich foci from EpiLCs at different time points of cellular induction (black, +24h n= 57 cells; orange, +48h, n= 48 cells; red, +72h n = 266 cells). ****, p = 2.2e-16; K-S test.E. Total cell extracts from naive ESCs and EpiLCs (+72h) analyzed by western blot with antiserum against H4K20me3. Short and long exposures of the signal are shown. Loading control was achieved by Ponceau staining.F. Left panel, mean distribution of the FRET efficiency related to the pixel fraction in nucleosome-rich foci from untreated EpiLCs (black, n = 124 cells) and A-196 EpiLCs treated cells (red, n = 113 cells). ****, p = 2.2e-16; K-S test. Right panel, Box-and-Whisker plots indicate the FRET efficiency (%) from untreated EpiLCs (black, n = 124 cells) and treated with A-196 during 2 days (red, n = 113 cells). The box plots indicate median values (middle lines), first and third quartiles (box edges) and the whiskers cover the minimum to maximum value range of the FRET efficiency.


Figure 1A . Total cell lysates from wild type ( WT ) and HAT1 knockout ( KO ) Clone 1 HepG2 cells were analyzed for the levels of Ksucc and Kac by Western blot analysis ( left ) . The expression levels of HAT1 , Histone H3 ( H3 ) , and beta - actin ( β - actin ) were measured by Western blot analysis in the cells ( right ) .

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Figure 1A. Total cell lysates from wild type (WT) and HAT1 knockout (KO) Clone 1 HepG2 cells were analyzed for the levels of Ksucc and Kac by Western blot analysis (left). The expression levels of HAT1, Histone H3 (H3), and beta-actin (β-actin) were measured by Western blot analysis in the cells (right).


Figure 2D . HAT1 - catalyzed histone H3 succinylation was analyzed by mixing purified HAT1 , histone H3 , and succinyl - CoA ( 2 μM ) with or without the addition of the indicated concentrations of CoA . Western blot analysis was performed with indicated antibodies . E . HAT1 - mediated histone H3 succinylation was assessed by mixing purified HAT1 , histone H3 , and succinyl - CoA ( 2 μM ) with or without acetyl - CoA ( 2 μM ) . Western blot analysis was performed with the indicated antibodies .

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Figure 2D. HAT1-catalyzed histone H3 succinylation was analyzed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies.E. HAT1-mediated histone H3 succinylation was assessed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without acetyl-CoA (2 μM). Western blot analysis was performed with the indicated antibodies.


Figure 3A . The levels of histone H3K122 succinylation , histone H3K122 acetylation , histone H3K27 acetylation , histone H3 , HAT1 , and β - actin were examined by Western blot analysis in HepG2 , HAT1 knockout ( KO ) Clone 1 HepG2 cells , and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 ( T188A ) . D . The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti - H3K122 succinylation antibody and anti - H3K122 acetylation antibody , and followed by quantitative PCR with primers of the promoter regions of CREBBP , BPTF and RPTOR in WT HepG2 cells , HAT1 KO HepG2 cells and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 ( T188A ) . N = 3 biological replicates . Data were presented as mean ± SD . Student ' s t - test , * * * P < 0 . 001 ; ns , no significance .

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Figure 3A. The levels of histone H3K122 succinylation, histone H3K122 acetylation, histone H3K27 acetylation, histone H3, HAT1, and β-actin were examined by Western blot analysis in HepG2, HAT1 knockout (KO) Clone 1 HepG2 cells, and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 (T188A).D. The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti-H3K122 succinylation antibody and anti-H3K122 acetylation antibody, and followed by quantitative PCR with primers of the promoter regions of CREBBP, BPTF and RPTOR in WT HepG2 cells, HAT1 KO HepG2 cells and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data were presented as mean ± SD. Student's t-test, ***P < 0.001; ns, no significance.


Figure 4B . Bar graphs depicting the relative ratio of succinylation in HAT1 KO versus WT of the 7 key enzymes in the glycolysis pathway depicted in ( A ) . N = 3 biological replicates . Data were presented as mean ± SD .

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Figure 4B. Bar graphs depicting the relative ratio of succinylation in HAT1 KO versus WT of the 7 key enzymes in the glycolysis pathway depicted in (A). N = 3 biological replicates. Data were presented as mean ± SD.


Figure 5D . HAT1 - catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1 , PGAM1 , and succinyl - CoA / acetyl - CoA in the in vitro succinylation assays . Western blot analysis was performed with indicated antibodies . F . The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2 - PG and 3 - PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant ( K99R ) . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - test , * * P < 0 . 01 .

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Figure 5D. HAT1-catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1, PGAM1, and succinyl-CoA/acetyl-CoA in the in vitro succinylation assays. Western blot analysis was performed with indicated antibodies.F. The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2-PG and 3-PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant (K99R). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.


Figure 6A . HAT1 levels were assessed by immunohistochemical staining in clinical tissues of liver cancer patients , pancreatic cancer patients , and cholangiocarcinoma patients . Scale bar = 100 μmB . The effect of HAT1 - mediated succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - test , * * P < 0 . 01 . C . The effect of PGAM1 succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - test , * * P < 0 . 01 . HepG2 cells , HAT1 KO HepG2 cells , and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 ( T188A ) were subcutaneously injected into athymic nude mice Tumor volumes and average tumor weight were calculated . N = 6 biological replicates . Scale bar = 10 mm . In the boxplots , boxes extend from the 25th to 75th percentiles ( inter ‐ quartile range ( IQR ) ) , central band represents the median , whiskers indicate the lowest and highest data within 1 . 5 × IQR from the lower and upper quartiles , respectively . Student ' s t - test , * * * P < 0 .

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Figure 6A. HAT1 levels were assessed by immunohistochemical staining in clinical tissues of liver cancer patients, pancreatic cancer patients, and cholangiocarcinoma patients. Scale bar = 100 μmB. The effect of HAT1-mediated succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. C. The effect of PGAM1 succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 (T188A) were subcutaneously injected into athymic nude mice Tumor volumes and average tumor weight were calculated. N = 6 biological replicates. Scale bar = 10 mm. In the boxplots, boxes extend from the 25th to 75th percentiles (inter‐quartile range (IQR)), central band represents the median, whiskers indicate the lowest and highest data within 1.5 × IQR from the lower and upper quartiles, respectively. Student's t-test, ***P < 0.001


Figure 2G ) Representative images of the EdU staining in freshly isolated YC and OC FAPs following EdU in vivo exposure for 3 days ( EdU in red / DAPI in blue ) . Scale bar = 25 μm . H ) Graph showing the percentage of EdU positive cells for the staining shown in ( G ) . I ) Representative images of the LMNB1 staining in freshly isolated YC and OC FAPs ( LMNB1 in green / DAPI in blue ) . Scale bar = 25 μm . J ) Graph showing the relative quantification of LMNB1 fluorescence intensity normalized for nuclei number , in the staining shown in ( I ) . L ) Representative images of the γH2AX and KI - 67 staining in freshly isolated YC and OC FAPs ( γH2AX in green / KI - 67 in red / DAPI in blue ) . Scale bar = 10 μm . Magnification in the insert ( scale bar = 3 μm ) shows γH2AX positive CCF in OC FAPs . M ) Graph showing the percentage of cells positive for γH2AX positive CCF , for the staining shown in ( L ) . N ) Graph showing the percentage of cells positive for KI - 67 for the staining shown in ( L ) .

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Figure 2G) Representative images of the EdU staining in freshly isolated YC and OC FAPs following EdU in vivo exposure for 3 days (EdU in red/DAPI in blue). Scale bar = 25 μm. H) Graph showing the percentage of EdU positive cells for the staining shown in (G).I) Representative images of the LMNB1 staining in freshly isolated YC and OC FAPs (LMNB1 in green/DAPI in blue). Scale bar = 25 μm. J) Graph showing the relative quantification of LMNB1 fluorescence intensity normalized for nuclei number,in the staining shown in (I).L) Representative images of the γH2AX and KI-67 staining in freshly isolated YC and OC FAPs (γH2AX in green/KI-67 in red/DAPI in blue). Scale bar = 10 μm. Magnification in the insert (scale bar = 3 μm) shows γH2AX positive CCF in OC FAPs. M) Graph showing the percentage of cells positive for γH2AX positive CCF, for the staining shown in (L). N) Graph showing the percentage of cells positive for KI-67 for the staining shown in (L).


Figure 4D ) Representative images of the phospho - NFkB staining in FAPs ( pNFkB - p65 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal section of YC , YT , OC and OT mice ; Scale bar = 25 μm ; E ) Box plot showing the percentage of pNFkB - p65 positive FAPs in the staining shown in ( D ) F ) Representative images of the phospho - p38 staining in FAPs ( pP38 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal sections of YC , YT , OC and OT mice . Scale bar = 25 μm ; G ) Box plot showing the percentage of pP38 positive FAPs in the staining shown inH ) Representative images of the phospho - SMAD2 / 3 staining in FAPs ( pSMAD2 / 3 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal sections of YC , YT , OC and OT mice . Scale bar = 25 μm ; I ) Box plot showing the percentage of pSMAD2 / 3 positive FAPs in the staining shown in ( H ) .

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Figure 4D) Representative images of the phospho-NFkB staining in FAPs (pNFkB-p65 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal section of YC, YT, OC and OT mice; Scale bar = 25 μm; E) Box plot showing the percentage of pNFkB-p65 positive FAPs in the staining shown in (D)F) Representative images of the phospho-p38 staining in FAPs (pP38 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; G) Box plot showing the percentage of pP38 positive FAPs in the staining shown inH) Representative images of the phospho-SMAD2/3 staining in FAPs (pSMAD2/3 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; I) Box plot showing the percentage of pSMAD2/3 positive FAPs in the staining shown in (H).


Figure 4 ( C ) Western blot analysis of cells transfected with 10 nM siRNA against ERK1 , ERK2 or a 5 nM combination of each ( ERK1 + 2 ) .

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Figure 4(C) Western blot analysis of cells transfected with 10 nM siRNA against ERK1, ERK2 or a 5 nM combination of each (ERK1+2).


Figure 1F , Representative immunoblot and quantification of pFAK levels in 5 % LPDS ( - LDL ) and upon increasing LDL loading times . Mean ± SD , n = 3 , paired student ' s t - test ( * p < 0 . 05 ) . G , Representative confocal images of NPC1 , internalized integrin β1 and pFAK localization . Stably NPC1 - GFP expressing cells were loaded with 2 μg / mL integrin β1 antibody for 30 min before fixation and stained with pFAK antibody followed by secondary antibodies . Dashed line indicates the cell edge . H , Quantification of G using Mander ' s colocalization for the indicated markers . Mean ± SD , n = 5 cells . Cells were treated with 5 % LPDS for 1 day and then without ( - ) or with ( + ) 50 μg / ml LDL for 1h .

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Figure 1F, Representative immunoblot and quantification of pFAK levels in 5% LPDS (-LDL) and upon increasing LDL loading times. Mean ± SD, n = 3, paired student's t-test (*p < 0.05).G, Representative confocal images of NPC1, internalized integrin β1 and pFAK localization. Stably NPC1-GFP expressing cells were loaded with 2 μg/mL integrin β1 antibody for 30 min before fixation and stained with pFAK antibody followed by secondary antibodies. Dashed line indicates the cell edge. H, Quantification of G using Mander's colocalization for the indicated markers. Mean ± SD, n = 5 cells. Cells were treated with 5 % LPDS for 1 day and then without (-) or with (+) 50 μg/ml LDL for 1h.


Figure 2D , After 1 day LDL depletion in 5 % LPDS , cells were loaded with 50 μg / ml LDL for the indicated times and intracellular cholesterol was detected with D4H as in C by confocal microscopy . CM , complete medium . Dashed lines indicate cell outlines . E , Quantification of D . Mean ± SD , n = 25 - 26 cells pooled from 2 independent experiments . F , Cells were treated for 1 day in 5 % LPDS followed by 50 μg / ml LDL loading for the indicated times . Representative confocal images of cells triple labeled with intracellular accessible cholesterol ( D4H ) , NPC1 - GFP and internalized integrin β1 . Dashed lines indicate cell outlines . G , Quantification of D4H area positive for NPC1 or internalized integrin β1 as in F . H , Fraction of D4H area overlapping with NPC1 or internalized integrin β1 in a peripheral region toward the leading edge ( exemplary box shown as inset in F ) . Mean ± SD , n = 27 - 30 cells pooled from 2 independent experiments . Student ' s t - test .

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Figure 2D, After 1 day LDL depletion in 5 % LPDS, cells were loaded with 50 μg/ml LDL for the indicated times and intracellular cholesterol was detected with D4H as in C by confocal microscopy. CM, complete medium. Dashed lines indicate cell outlines. E, Quantification of D. Mean ± SD, n = 25-26 cells pooled from 2 independent experiments. F, Cells were treated for 1 day in 5 % LPDS followed by 50 μg/ml LDL loading for the indicated times. Representative confocal images of cells triple labeled with intracellular accessible cholesterol (D4H), NPC1-GFP and internalized integrin β1. Dashed lines indicate cell outlines. G, Quantification of D4H area positive for NPC1 or internalized integrin β1 as in F. H, Fraction of D4H area overlapping with NPC1 or internalized integrin β1 in a peripheral region toward the leading edge (exemplary box shown as inset in F). Mean ± SD, n = 27-30 cells pooled from 2 independent experiments. Student's t-test.


Figure 3A , After 1 day in 5 % LPDS , NPC1 - GFP expressing cells were loaded with 50 μg / ml LDL with or without 10 μM PF228 for the indicated times , and stained for intracellular D4H . Representative confocal images of intracellular D4H , NPC1 - GFP and internalized integrin β1 labeling . Dashed lines indicate cell outlines . B , Fraction of D4H positive area in the cell as in A . C , Fraction of D4H area overlapping with NPC1 in a peripheral region toward the leading edge ( exemplary box shown as inset in A ) . D , Quantification of D4H area positive for integrin β1 in a peripheral region toward the leading edge . Mean ± SD , n = 26 - 30 cells pooled from 2 independent experiments . Student ' s t - test . I , After 1 day 5 % LPDS , cells were loaded with 50 μg / ml LDL with or without 10 μM PF228 for the indicated times and endomembrane PI ( 4 , 5 ) P2 was stained with antibody and imaged by confocal microscopy . J , Quantification of mean PI ( 4 , 5 ) P2 immunoreactivity in cells . Mean ± SD , n = 16 - 26 cells pooled from 2 independent experiments . Student ' s t - 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 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

Figure 3A, After 1 day in 5 % LPDS, NPC1-GFP expressing cells were loaded with 50 μg/ml LDL with or without 10 μM PF228 for the indicated times, and stained for intracellular D4H. Representative confocal images of intracellular D4H, NPC1-GFP and internalized integrin β1 labeling. Dashed lines indicate cell outlines. B, Fraction of D4H positive area in the cell as in A. C, Fraction of D4H area overlapping with NPC1 in a peripheral region toward the leading edge (exemplary box shown as inset in A). D, Quantification of D4H area positive for integrin β1 in a peripheral region toward the leading edge. Mean ± SD, n = 26-30 cells pooled from 2 independent experiments. Student's t-test. I, After 1 day 5 % LPDS, cells were loaded with 50 μg/ml LDL with or without 10 μM PF228 for the indicated times and endomembrane PI(4,5)P2 was stained with antibody and imaged by confocal microscopy.J, Quantification of mean PI(4,5)P2 immunoreactivity in cells. Mean ± SD, n = 16-26 cells pooled from 2 independent experiments. Student's t-test.


Figure 4C , Confocal image of endo - GFP - ORP2 and pFAK immunofluorescence staining in saponin pre - permeabilized cells loaded with LDL for 2 h before fixation . GFP antibody was used to enhance endo - GFP - ORP2 signal . Insets show examples of " no contact " , " contact " and " overlap " of organelles . Dashed line indicates cell edge . D , Quantification of pFAK , integrin β1 , and NPC1 association with endo - GFP - ORP2 in C . n = 380 pFAK ; 353 integrin β1 ; 232 NPC1 organelles in 2 cells . F , Schematic outline for plasma membrane D4H labeling in combination with ORP2 degron system , and representative widefield epifluorescent images of co - plated cells after 4 h of LDL loading . Asterisk indicates ORP2 - depleted degron cells . Dashed lines separate ORP2 expressing and depleted cells . G , Quantification of F . Mean ± SD , n = 20 fields of cells pooled from 2 independent experiments . Student ' s t - 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 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

Figure 4C, Confocal image of endo-GFP-ORP2 and pFAK immunofluorescence staining in saponin pre-permeabilized cells loaded with LDL for 2 h before fixation. GFP antibody was used to enhance endo-GFP-ORP2 signal. Insets show examples of "no contact", "contact" and "overlap" of organelles. Dashed line indicates cell edge. D, Quantification of pFAK, integrin β1, and NPC1 association with endo-GFP-ORP2 in C. n = 380 pFAK; 353 integrin β1; 232 NPC1 organelles in 2 cells. F, Schematic outline for plasma membrane D4H labeling in combination with ORP2 degron system, and representative widefield epifluorescent images of co-plated cells after 4 h of LDL loading. Asterisk indicates ORP2-depleted degron cells. Dashed lines separate ORP2 expressing and depleted cells. G, Quantification of F. Mean ± SD, n = 20 fields of cells pooled from 2 independent experiments. Student's t-test.