( E ) Box plot showing the distribution of ΔF / F0 across multiple neurons on the basal condition , on NMDA treatment in the presence Ca2 + , on NMDA treatment in the absence of Ca2 + , on NMDA treatment with D - AP5 and on NMDA treatment along with MK - 801 . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n ≥ 30 neurons per group from 3 independent platings .

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(E) Box plot showing the distribution of ΔF/F0 across multiple neurons on the basal condition, on NMDA treatment in the presence Ca2+, on NMDA treatment in the absence of Ca2+, on NMDA treatment with D-AP5 and on NMDA treatment along with MK-801. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n≥30 neurons per group from 3 independent platings.


( B ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( pseudo - colored ) in cortical neurons on the basal condition and on DHPG ( 50μM ) treatment for 5 minutes in the presence or absence of AICAR ( 1mM ) . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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(B) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (pseudo-colored) in cortical neurons on the basal condition and on DHPG (50μM) treatment for 5 minutes in the presence or absence of AICAR (1mM). MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( C ) Box plot showing the normalized phospho - eEF2 intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 17 - 46 cells per group from 3 independent platings .

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(C) Box plot showing the normalized phospho-eEF2 intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=17-46 cells per group from 3 independent platings.


( D ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes , on AICAR treatment and on DHPG treatment with AICAR . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * * * * p < 0 . 0001 , n = 18 - 57 cells per group from 3 independent platings .

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(D) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. ****p<0.0001, n=18-57 cells per group from 3 independent platings.


( E ) Representative images showing the phospho - eEF2 immunolabeling ( Pseudo - colored ) and newly synthesized proteins as FUNCAT signal ( Pseudo - colored ) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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(E) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (Pseudo-colored) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( F ) Box plot showing phospho - eEF2 intensity distribution across multiple neurons on basal condition ( Vehicle control ) , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 21 - 36 cells per group from 3 independent platings .

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(F) Box plot showing phospho-eEF2 intensity distribution across multiple neurons on basal condition (Vehicle control), on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=21-36 cells per group from 3 independent platings.


( G ) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition , on NMDA treatment for 5 minutes , on Compound C treatment and on NMDA treatment with Compound C . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 24 - 52 cells per group from 3 independent platings .

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

(G) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, ****p<0.0001, n=24-52 cells per group from 3 independent platings.


Freezing phenotype ( A ) of Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA for non - acclimation , − 5 ° C , 30 min ; CA for cold acclimation , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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

Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) , and ion leakage ( C ) of Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA for non - acclimation , − 5 ° C , 30 min ; CA for cold acclimation , − 8 ° C , 40 min ) .

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survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min).


Expression of CBFs ( D ) in Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were placed at 4 ° C for 3 h ( D ) and total RNA was extracted and subjected to qRT - PCR analysis . Relative transcript levels in untreated Col - 0 plants were set to 1 .

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Expression of CBFs (D) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 3 h (D) and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.


Expression of CBFs targets ( E ) in Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were placed at 4 ° C for 24 h ( E ) , and total RNA was extracted and subjected to qRT - PCR analysis . Relative transcript levels in untreated Col - 0 plants were set to 1 .

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Expression of CBFs targets (E) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 24 h (E), and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.


A Time - course analysis of cytosolic free calcium concentration ( [ Ca2 + ] cyt ) dynamics in 10 - d - old wild type ( Col - 0 ) , atann1 , and atann1 AtANN1 complementation line ( # 10 ) after treatment with ice - cold water or ice - cold water containing 10 mM LaCl3 ( arrow shows the time point of treatment ) . Luminescence was recorded at 1 - s intervals . B Quantification of the cold - induced [ Ca2 + ] cyt changes shown in ( A ) . Peak [ Ca2 + ] cyt indicates the highest [ Ca2 + ] cyt after treatment .

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A Time-course analysis of cytosolic free calcium concentration ([Ca2+]cyt) dynamics in 10-d-old wild type (Col-0), atann1, and atann1 AtANN1 complementation line (#10) after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.


C GST pull - down assay showing that OST1 interacts with AtANN1 in vitro . Purified recombinant GST - OST1 , GST - OST1G33R , or GST proteins from E . coli were immunoprecipitated with GST beads and then incubated with MBP - His - AtANN1 . Precipitated proteins were detected with anti - GST and anti - His antibodies .

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C GST pull-down assay showing that OST1 interacts with AtANN1 in vitro. Purified recombinant GST-OST1, GST-OST1G33R, or GST proteins from E. coli were immunoprecipitated with GST beads and then incubated with MBP-His-AtANN1. Precipitated proteins were detected with anti-GST and anti-His antibodies.


D Interaction of OST1 and AtANN1 detected by bimolecular fluorescence complementation ( BiFC ) assays . The construct combinations were co - transformed into N . benthamiana leaves and expressed for 2 d . The signal was detected by confocal microscopy . Scale bar , 25 µm . The fluorescence intensity was scanned using the ImageJ plot profile tool . The y - axes indicate relative pixel intensity .

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D Interaction of OST1 and AtANN1 detected by bimolecular fluorescence complementation (BiFC) assays. The construct combinations were co-transformed into N. benthamiana leaves and expressed for 2 d. The signal was detected by confocal microscopy. Scale bar, 25 µm. The fluorescence intensity was scanned using the ImageJ plot profile tool. The y-axes indicate relative pixel intensity.


E , F Interaction of OST1 and AtANN1 detected by Co - IP assays in Arabidopsis . Twelve - day - old OST1 - Myc overexpressing plants grown on MS medium at 22 ° C were placed at 4 ° C for 0 , 0 . 5 , 2 h , and total proteins were extracted and immunoprecipitated with anti - Myc agarose beads . The wild type ( Col - 0 ) treated with 4 ° C for 2 h was used as control . The OST1 - Myc protein was detected with anti - Myc antibody and the AtANN1 protein was detected with anti - AtANN1 antibody . Representative pictures are shown in ( E ) and relative protein level in ( F ) .

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E,F Interaction of OST1 and AtANN1 detected by Co-IP assays in Arabidopsis. Twelve-day-old OST1-Myc overexpressing plants grown on MS medium at 22°C were placed at 4°C for 0, 0.5, 2 h, and total proteins were extracted and immunoprecipitated with anti-Myc agarose beads. The wild type (Col-0) treated with 4°C for 2 h was used as control. The OST1-Myc protein was detected with anti-Myc antibody and the AtANN1 protein was detected with anti-AtANN1 antibody. Representative pictures are shown in (E) and relative protein level in (F).


G Co - IP assay showing the interaction between OST1 and AtANN1 in vivo . The construct combinations were expressed in N . benthamiana leaves . Total proteins were extracted after the N . benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti - Myc agarose beads . The proteins were detected with anti - Myc and anti - GFP antibodies .

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G Co-IP assay showing the interaction between OST1 and AtANN1 in vivo. The construct combinations were expressed in N. benthamiana leaves. Total proteins were extracted after the N. benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti-Myc agarose beads. The proteins were detected with anti-Myc and anti-GFP antibodies.


Freezing phenotype ( A ) of Col - 0 , ost1 - 3 , atann1 , and atann1 ost1 - 3 . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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Freezing phenotype (A) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) and ion leakage ( C ) of Col - 0 , ost1 - 3 , atann1 , and atann1 ost1 - 3 . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

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survival rate (B) and ion leakage (C) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


OST1 gene expression D ) in Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 .

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OST1 gene expression D) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1.


protein levels of OST1 and AtANN1 ( E ) in Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . OST1 and AtANN1 proteins were detected with anti - Myc and anti - AtANN1 antibodies . HSP90 was used as a control .

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protein levels of OST1 and AtANN1 (E) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1. OST1 and AtANN1 proteins were detected with anti-Myc and anti-AtANN1 antibodies. HSP90 was used as a control.


Freezing phenotype ( F ) of Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , - 5 ° C , 30 min ; CA , - 8 ° C , 40 min ) . Representative pictures are shown in ( F ) .

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Freezing phenotype (F) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min). Representative pictures are shown in (F).


survival rate ( G ) , and ion leakage ( H ) of Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , - 5 ° C , 30 min ; CA , - 8 ° C , 40 min ) .

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survival rate (G), and ion leakage (H) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min).


A , B OST1 phosphorylates AtANN1 ( A ) and AtANN4 ( B ) in vitro . Purified recombinant MBP - His - OST1 was incubated with GST - AtANN1 , GST - AtANN4 or GST in kinase reaction buffer with 1 µCi [ γ - 32P ] ATP for 30 min at 30 ° C , followed by separation with SDS - PAGE . Phosphorylated AtANN1 and AtANN4 were detected by autoradiography . Recombinant OST1 , AtANN1 and AtANN4 were stained by Coomassie brilliant blue ( CBB ) .

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A,B OST1 phosphorylates AtANN1 (A) and AtANN4 (B) in vitro. Purified recombinant MBP-His-OST1 was incubated with GST-AtANN1, GST-AtANN4 or GST in kinase reaction buffer with 1 µCi [γ-32P] ATP for 30 min at 30°C, followed by separation with SDS-PAGE. Phosphorylated AtANN1 and AtANN4 were detected by autoradiography. Recombinant OST1, AtANN1 and AtANN4 were stained by Coomassie brilliant blue (CBB).


C In - gel kinase assays of OST1 in Col - 0 and ost1 - 3 mutant under cold stress . Twelve - day - old Col - 0 and ost1 - 3 mutant were treated at 4 ° C for 2 h . Total protein extracts were prepared and separated on a SDS - PAGE gel containing 0 . 2 mg / mL GST - AtANN1 as a substrate , and incubated with 70 µCi [ γ - 32P ] ATP . Top , autoradiograph ; bottom , CBB staining .

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C In-gel kinase assays of OST1 in Col-0 and ost1-3 mutant under cold stress. Twelve-day-old Col-0 and ost1-3 mutant were treated at 4°C for 2 h. Total protein extracts were prepared and separated on a SDS-PAGE gel containing 0.2 mg/mL GST-AtANN1 as a substrate, and incubated with 70 µCi [γ-32P] ATP. Top, autoradiograph; bottom, CBB staining.


D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1 . After phosphorylation , the proteins were separated by SDS - PAGE and subjected to autoradiography . Top , autoradiograph ; bottom , CBB staining .

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D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1. After phosphorylation, the proteins were separated by SDS-PAGE and subjected to autoradiography. Top, autoradiograph; bottom, CBB staining.


E OST1 phosphorylates AtANN1 at S289 in vivo in LC / MS analysis . Total proteins were extracted from 12 - d - old AtANN1 - Myc overexpressing plants treated at 4 ° C for 0 , 10 , 30 and 120 min , followed by trypsin digestion . Phosphopeptides were enriched for mass spectrometry analysis . Representative picture for 10 min was shown .

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E OST1 phosphorylates AtANN1 at S289 in vivo in LC/MS analysis. Total proteins were extracted from 12-d-old AtANN1-Myc overexpressing plants treated at 4°C for 0, 10, 30 and 120 min, followed by trypsin digestion. Phosphopeptides were enriched for mass spectrometry analysis. Representative picture for 10 min was shown.


Freezing phenotype ( A ) of Col - 0 , atann1 , and atann1 AtANN1S289E ( # 5 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1S289E (#5) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) , and ion leakage ( C ) of Col - 0 , atann1 , and atann1 AtANN1S289E ( # 5 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

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survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1S289E (#5) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


Freezing phenotype ( D ) of Col - 0 , Atann1 , and atann1 AtANN1S289A ( # 16 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( D ) .

Freezing phenotype (D) of Col-0, Atann1, and atann1 AtANN1S289A (#16) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (D).


survival rate ( E ) , and ion leakage ( F ) of Col - 0 , Atann1 , and atann1 AtANN1S289A ( # 16 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

survival rate (E), and ion leakage (F) of Col-0, Atann1, and atann1 AtANN1S289A (#16) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


A AtANN1 current recordings in Xenopus oocytes . Whole - cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1 , OST1 , or AtANN1 + OST1 cRNA . Bath solution was described in Materials and Methods . The voltage protocol as well as time and current scale bars for the recordings are shown . B Current - voltage ( I - V ) relationship of the steady - state whole - cell currents in Xenopus oocytes described in ( A ) . Negative current is influx of cations into the oocytes .

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

A AtANN1 current recordings in Xenopus oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, OST1, or AtANN1 + OST1 cRNA. Bath solution was described in Materials and Methods. The voltage protocol as well as time and current scale bars for the recordings are shown. B Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (A). Negative current is influx of cations into the oocytes.


C Percentage of total Xenophus oocytes in different nA range according to ( A ) .

C Percentage of total Xenophus oocytes in different nA range according to (A).


D AtANN1 current recordings in oocytes . Whole - cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1 , AtANN1S289A , OST1 , AtANN1 + OST1 , and AtANN1S289A + OST1 cRNA . E Current - voltage ( I - V ) relationship of the steady - state whole - cell currents in Xenopus oocytes described in ( D ) .

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

D AtANN1 current recordings in oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, AtANN1S289A, OST1, AtANN1 + OST1, and AtANN1S289A + OST1 cRNA. E Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (D).


F Percentage of total Xenophus oocytes in different nA range according to ( D ) .

F Percentage of total Xenophus oocytes in different nA range according to (D).


A Time - course analysis of [ Ca2 + ] cyt dynamics between 10 - day - old wild type ( Col - 0 ) and ost1 - 3 mutant after treatment with ice - cold water or ice - cold water containing 10 mM LaCl3 ( arrow shows the time point of treatment ) . Luminescence was recorded at 1 - s intervals . B Quantification of the cold - induced [ Ca2 + ] cyt changes shown in ( A ) . Peak [ Ca2 + ] cyt indicates the highest [ Ca2 + ] cyt after treatment .

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

A Time-course analysis of [Ca2+]cyt dynamics between 10-day-old wild type (Col-0) and ost1-3 mutant after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.


C MST assays of the calcium - binding affinity of GST - AtANN1 , GST - AtANN1S289A , and GST - AtANN1S289E . GST was used as a control .

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

C MST assays of the calcium-binding affinity of GST-AtANN1, GST-AtANN1S289A, and GST-AtANN1S289E. GST was used as a control.


A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours ( n = 4 , 632 tumours in each group ) across TCGA . - log10 ( padj ) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y - axis . Signatures more highly represented in TERThigh tumours are shown on the right , while those in TERTlow tumours are shown on the left .

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

A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours (n = 4,632 tumours in each group) across TCGA. -log10(padj) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y-axis. Signatures more highly represented in TERThigh tumours are shown on the right, while those in TERTlow tumours are shown on the left.


D - F RT - qPCR analysis of interferon - related genes ( D ) , western blot analysis of TERT , TBK1 , pTBK1 , IRF3 , pIRF3 , and GAPDH levels ( E ) , and ELISA of IFNβ and CXCL10 in culture supernatant ( F ) in U2OS and HeLa cells ectopically expressing TERT - WT or TERT - K626A .

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

D-F RT-qPCR analysis of interferon-related genes (D), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (E), and ELISA of IFNβ and CXCL10 in culture supernatant (F) in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A.


D RT - qPCR analysis of TA - ERVs in U2OS and HeLa cells ectopically expressing TERT - WT or TERT - K626A . E RT - qPCR analysis of TA - ERVs in HeLa and HCT116 cells with TERT knockdown .

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D RT-qPCR analysis of TA-ERVs in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A. E RT-qPCR analysis of TA-ERVs in HeLa and HCT116 cells with TERT knockdown. F


F - H RT - qPCR analysis of interferon - related genes and TA - ERVs ( F ) , western blot analysis of TERT , TBK1 , pTBK1 , IRF3 , pIRF3 , and GAPDH levels ( G ) , and ELISA of IFNβ and CXCL10 in culture supernatant ( H ) in U2OS cells ectopically expressing TERT - WT or TERT - K626A using Dox - inducible system . GFP was used as negative control . Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH , as indicated at the bottom of the blot ( G ) .

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

F-H RT-qPCR analysis of interferon-related genes and TA-ERVs (F), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (G), and ELISA of IFNβ and CXCL10 in culture supernatant (H) in U2OS cells ectopically expressing TERT-WT or TERT-K626A using Dox-inducible system. GFP was used as negative control. Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH, as indicated at the bottom of the blot (G).


D MeDIP - qPCR analysis of TA - ERVs sites in HeLa cells ectopically expressing TERT - WT or TERT - K626A . Primer sets recognizing exon 8 of the human ZC3H13 gene and a gene desert on chromosome 12 were used for positive and negative controls , respectively .

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

D MeDIP-qPCR analysis of TA-ERVs sites in HeLa cells ectopically expressing TERT-WT or TERT-K626A. Primer sets recognizing exon 8 of the human ZC3H13 gene and a gene desert on chromosome 12 were used for positive and negative controls, respectively.


E ChIP - qPCR for TERT and Sp1 targets on TA - ERVs in U2OS cells with TERT ectopic expression and in HeLa cells . GAPDH promoter was used as a negative control , and VEGF promoter was used as a positive control .

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

E ChIP-qPCR for TERT and Sp1 targets on TA-ERVs in U2OS cells with TERT ectopic expression and in HeLa cells. GAPDH promoter was used as a negative control, and VEGF promoter was used as a positive control.


A Expression of murine ERVs in blood of WT and G1 Tert - / - mice . n = 5 mice per group . B Expression of MMERGLN _ LTR and MMERGLN - int in livers of WT and G1 Tert - / - mice treated with ENU or saline ( control ) . n = 3 mice per group .

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A Expression of murine ERVs in blood of WT and G1 Tert-/- mice. n = 5 mice per group. B Expression of MMERGLN_LTR and MMERGLN-int in livers of WT and G1 Tert-/- mice treated with ENU or saline (control). n = 3 mice per group. C


D Heat map showing log2 fold change differences of interferon - related genes expression between ENU treatment and control ( saline ) in WT and G1 Tert - / - mice by RNA - seq . n = 3 mice per group . E Heat map showing log2 fold change differences of cytokine / chemokine between ENU treatment and control ( saline ) in WT and G1 Tert - / - mice by cytokine / chemokine array . n = 5 mice per group .

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D Heat map showing log2 fold change differences of interferon-related genes expression between ENU treatment and control (saline) in WT and G1 Tert-/- mice by RNA-seq. n = 3 mice per group. E Heat map showing log2 fold change differences of cytokine/chemokine between ENU treatment and control (saline) in WT and G1 Tert-/- mice by cytokine/chemokine array. n = 5 mice per group. F


B TCGA fragments per kilobase million ( FPKM ) values of TERT , CXCL10 , ISG15 , and OASL in tumours grouped in TERThigh ( n = 500 ) and TERTlow groups ( n = 500 ) . Data represent mean ± SD . P - values were analysed using unpaired two - tailed Student ' s t - tests .

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

B TCGA fragments per kilobase million (FPKM) values of TERT, CXCL10, ISG15, and OASL in tumours grouped in TERThigh (n = 500) and TERTlow groups (n = 500). Data represent mean ± SD. P-values were analysed using unpaired two-tailed Student's t-tests.


C TCGA FPKM values of interferon - related genes in tumours grouped in TERThigh ( n = 500 ) and TERTlow ( n = 500 ) groups . Heat map showing mean FPKM values of each gene .

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

C TCGA FPKM values of interferon-related genes in tumours grouped in TERThigh (n = 500) and TERTlow (n = 500) groups. Heat map showing mean FPKM values of each gene.


Expression of TERT ( D ) in TERThigh and TERTlow cancer types ( 6 types in each ) . ACC ( adrenocortical carcinoma ) , n = 79 . KICH ( kidney chromophobe ) , n = 66 . KIRP ( kidney renal papillary cell carcinoma ) , n = 291 . LGG ( brain lower grade glioma ) , n = 532 . PRAD , n = 502 . THCA , n = 513 . BLCA ( bladder urothelial carcinoma ) , n = 414 . CESC ( cervical squamous cell carcinoma and endocervical adenocarcinoma ) , n = 306 . DLBC ( lymphoid neoplasm diffuse large B - cell lymphoma ) , n = 48 . LUSC ( lung squamous cell carcinoma ) , n = 502 . OV ( ovarian serous cystadenocarcinoma ) , n = 430 . All boxplots include the median line , box indicates the interquartile range ( IQR ) and whiskers denote the 1 . 5 × IQR . P - values were analysed using two - tailed Mann - Whitney U test .

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

Expression of TERT (D) in TERThigh and TERTlow cancer types (6 types in each). ACC (adrenocortical carcinoma), n = 79. KICH (kidney chromophobe), n = 66. KIRP (kidney renal papillary cell carcinoma), n = 291. LGG (brain lower grade glioma), n = 532. PRAD, n = 502. THCA, n = 513. BLCA (bladder urothelial carcinoma), n = 414. CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), n = 306. DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), n = 48. LUSC (lung squamous cell carcinoma), n = 502. OV (ovarian serous cystadenocarcinoma), n = 430. All boxplots include the median line, box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. P-values were analysed using two-tailed Mann-Whitney U test.


Expression of DDX58 , IFIH1 , CXCL10 , IFI44 , ISG15 , and OASL ( E ) in TERThigh and TERTlow cancer types ( 6 types in each ) . ACC ( adrenocortical carcinoma ) , n = 79 . KICH ( kidney chromophobe ) , n = 66 . KIRP ( kidney renal papillary cell carcinoma ) , n = 291 . LGG ( brain lower grade glioma ) , n = 532 . PRAD , n = 502 . THCA , n = 513 . BLCA ( bladder urothelial carcinoma ) , n = 414 . CESC ( cervical squamous cell carcinoma and endocervical adenocarcinoma ) , n = 306 . DLBC ( lymphoid neoplasm diffuse large B - cell lymphoma ) , n = 48 . LUSC ( lung squamous cell carcinoma ) , n = 502 . OV ( ovarian serous cystadenocarcinoma ) , n = 430 . All boxplots include the median line , box indicates the interquartile range ( IQR ) and whiskers denote the 1 . 5 × IQR . P - values were analysed using two - tailed Mann - Whitney U test .

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

Expression of DDX58, IFIH1, CXCL10, IFI44, ISG15, and OASL (E) in TERThigh and TERTlow cancer types (6 types in each). ACC (adrenocortical carcinoma), n = 79. KICH (kidney chromophobe), n = 66. KIRP (kidney renal papillary cell carcinoma), n = 291. LGG (brain lower grade glioma), n = 532. PRAD, n = 502. THCA, n = 513. BLCA (bladder urothelial carcinoma), n = 414. CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), n = 306. DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), n = 48. LUSC (lung squamous cell carcinoma), n = 502. OV (ovarian serous cystadenocarcinoma), n = 430. All boxplots include the median line, box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. P-values were analysed using two-tailed Mann-Whitney U test.


G Representative sections of immunohistochemical staining of CD4 and FOXP3 in TERT - ERVshigh and TERT - ERVslow colon tumours . Red arrowheads indicate FOXP3 + cells . Scale bar = 100 μm .

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G Representative sections of immunohistochemical staining of CD4 and FOXP3 in TERT-ERVshigh and TERT-ERVslow colon tumours. Red arrowheads indicate FOXP3+ cells. Scale bar = 100 μm.


Chaining indices of the indicated genotypes . Statistical significance was compared with control . ; n = 9 , 6 , 7 ( E ) groups ( 10 flies per group ) . Courtship indices for one target ( Canton - S ) male and one tester male of the indicated genotypes . Changes were determined by comparing the data with control . ; n = 9 , 8 , 9 ( F ) flies . Sexual preference of single male of indicated genotypes toward a decapitated virgin female and a decapitated naïve male . Statistical significance was compared with the other gender . ; n = 10 , 10 , 10 ( F ) flies .

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

Chaining indices of the indicated genotypes. Statistical significance was compared with control. ; n = 9, 6, 7 (E) groups (10 flies per group). Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with control. ; n = 9, 8, 9 (F) flies. Sexual preference of single male of indicated genotypes toward a decapitated virgin female and a decapitated naïve male. Statistical significance was compared with the other gender. ; n = 10, 10, 10 (F) flies.


Courtship indices for one target ( Canton - S ) male and one tester male of the indicated genotypes . Changes were determined by comparing the data with relative control . n = 8 , 13 , 13 ( D )

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Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with relative control. n = 8, 13, 13 (D)


( A - C ) DA level ( pictograms per head ) in males of the indicated genotypes . n = 3 , 3 , 3 ( A ) ; n = 4 , 5 , 3 , 3 ( B ) ; n = 3 , 3 , 3 ( C ) times ( 100 heads per genotype ) .

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(A-C) DA level (pictograms per head) in males of the indicated genotypes. n = 3, 3, 3 (A); n = 4, 5, 3, 3 (B); n = 3, 3, 3 (C) times (100 heads per genotype).


( D ) Posterior confocal sections of UAS - mCD8 - GFP ; Ddc - Gal4 adult male brain in combination with UAS - LacZ or UAS - Myc . RNAi - 1 stained with antibody to nc82 ( pan - neuropil marker ; magenta ) . Scale bars represent 50 μm .

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(D) Posterior confocal sections of UAS-mCD8-GFP; Ddc-Gal4 adult male brain in combination with UAS-LacZ or UAS-Myc.RNAi-1 stained with antibody to nc82 (pan-neuropil marker; magenta). Scale bars represent 50 μm.


( F , G ) Western blot of proteins extracted from adult heads of indicated genotypes . Statistical analysis showing the protein level of GFP ( F ) or Ddc ( G ) to that of β - actin in Western blot .

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(F, G) Western blot of proteins extracted from adult heads of indicated genotypes. Statistical analysis showing the protein level of GFP (F) or Ddc (G) to that of β-actin in Western blot.


( B ) Drosophila S2 cells transfected with the pUAST - HA empty vector or the pUAST - HA - Myc plasmid were co - transfected with each of the firefly luciferase reporter vector bearing the fragment shown in ( A ) . After 48 hours , the luciferase activity was detected in each cell lysate ( n = 5 times ) .

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

(B) Drosophila S2 cells transfected with the pUAST-HA empty vector or the pUAST-HA-Myc plasmid were co-transfected with each of the firefly luciferase reporter vector bearing the fragment shown in (A). After 48 hours, the luciferase activity was detected in each cell lysate (n = 5 times).


( C ) Drosophila S2 cells transfected with the pUAST - HA - Myc plasmid or the empty vector were used for quantification of ChIP - PCRs ( n = 4 times ) .

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(C) Drosophila S2 cells transfected with the pUAST-HA-Myc plasmid or the empty vector were used for quantification of ChIP-PCRs (n = 4 times).


( D ) RNA from adult male heads ( n = 50 heads per genotype ) of indicated genotypes was subjected to real - time qPCR .

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(D) RNA from adult male heads (n = 50 heads per genotype) of indicated genotypes was subjected to real-time qPCR.


( E ) DA level ( pictograms per head ) in the head of males of the indicated genotypes . n = 4 ( 100 heads per genotype ) .

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

(E) DA level (pictograms per head) in the head of males of the indicated genotypes. n = 4 (100 heads per genotype).


B . Deconvolved fluorescence micrographs of Chm7 - GFP and truncations with Nup170 - mCherry NE marker and merge of green and red channels . Scale bar is 5 μm .

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B. Deconvolved fluorescence micrographs of Chm7-GFP and truncations with Nup170-mCherry NE marker and merge of green and red channels. Scale bar is 5 μm.


C . Scatter plot of the mean fluorescence intensity with SD of individual NE foci ( horizontal line is mean ) . > 50 foci were measured for each GFP fusion ; p values from un - paired student ' s T - test . * * * is p ≤ 0 . 001 ; * * * * is p ≤ 0 . 0001 .

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C. Scatter plot of the mean fluorescence intensity with SD of individual NE foci (horizontal line is mean). >50 foci were measured for each GFP fusion; p values from un-paired student's T-test. *** is p ≤ 0.001; **** is p ≤ 0.0001.


D . Deconvolved fluorescence micrographs of Chm7 - GFP and Mps3 - mCherry with merge of green and red images ( right ) . Scale bar is 5 μm .

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D. Deconvolved fluorescence micrographs of Chm7-GFP and Mps3-mCherry with merge of green and red images (right). Scale bar is 5 μm.


E . 10 - fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid ( - ) or those expressing the indicated Chm7 constructs ( pRS416 - HA - CHM7 , pRS416 - HA - chm7 - NTD , pRS416 - HA - chm7 - CTD ) . See Appendix Figure S2A for protein levels .

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E. 10-fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid (-) or those expressing the indicated Chm7 constructs (pRS416-HA-CHM7, pRS416-HA-chm7-NTD, pRS416-HA-chm7-CTD). See Appendix Figure S2A for protein levels.


A . Deconvolved inverted fluorescence micrographs Chm7 - GFP and truncations in the indicated null strains . Scale bar is 5 μm . B . Plot of the percentage of cells with SD of Chm7 - GFP ( and truncations ) NE foci from A . Data are from 3 independent replicates where > 200 cells were counted for each strain . p values from un - paired student ' s T - test . * * is a p value ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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A. Deconvolved inverted fluorescence micrographs Chm7-GFP and truncations in the indicated null strains. Scale bar is 5 μm.B. Plot of the percentage of cells with SD of Chm7-GFP (and truncations) NE foci from A. Data are from 3 independent replicates where >200 cells were counted for each strain. p values from un-paired student's T-test. ** is a p value ≤ 0.01; ****, p ≤ 0.0001.


C . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in a heh1Δ strain containing either an empty pRS426 plasmid ( - ) or those expressing HEH1 ( pRS426 - HEH1 ) or HEH2 ( pRS426 - HEH2 ) . D . Plots of the percentage of cells from C with Chm7 - GFP NE - foci . Data are from 3 independent replicates where > 200 cells were counted for each strain . Error bars are SD . p values from T - test . * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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C. Deconvolved inverted fluorescence micrographs of Chm7-GFP in a heh1Δ strain containing either an empty pRS426 plasmid (-) or those expressing HEH1 (pRS426-HEH1) or HEH2 (pRS426-HEH2).D. Plots of the percentage of cells from C with Chm7-GFP NE-foci. Data are from 3 independent replicates where >200 cells were counted for each strain. Error bars are SD. p values from T-test. **, p ≤ 0.01; ****, p ≤ 0.0001.


C . GST , GST - Snf7 and GST - snf7OPEN were immobilized on GT - resin and incubated with buffer ( - ) or recombinant heh2 ( 1 - 308 ) - His6 . Bound proteins separated by SDS - PAGE were visualized by coomassie stain and by Western blot with anti - His6 antibody and ECL detection ( WB ; top panel ) . Middle panel shows indicated cropped region of gel where contrast has been increased . Numbers on side of gel show position of molecular weight ( MW ) markers .

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C. GST, GST-Snf7 and GST-snf7OPEN were immobilized on GT-resin and incubated with buffer(-) or recombinant heh2(1-308)-His6. Bound proteins separated by SDS-PAGE were visualized by coomassie stain and by Western blot with anti-His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of gel where contrast has been increased. Numbers on side of gel show position of molecular weight (MW) markers.


D . In vitro transcription translation ( IVT ) reactions generating radiolabeled ( 35S ) truncations of Heh2 ( inputs ) were incubated with bead - bound GST , GST - snf7OPEN or GST - Snf7 before washing , elution and detection of bound proteins by autoradiography ( top panel ) or coomassie staining ( bottom ) .

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D. In vitro transcription translation (IVT) reactions generating radiolabeled (35S) truncations of Heh2 (inputs) were incubated with bead-bound GST, GST-snf7OPEN or GST-Snf7 before washing, elution and detection of bound proteins by autoradiography (top panel) or coomassie staining (bottom).


E . As in C except with GST - Chm7 constructs . Upper panel shows western blot ( WB ) with anti - His6 antibody . * chm7 - NTD is also expressed with a His6 tag . Bottom panel is coomassie stained .

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E. As in C except with GST-Chm7 constructs. Upper panel shows western blot (WB) with anti-His6 antibody. *chm7-NTD is also expressed with a His6 tag. Bottom panel is coomassie stained.


A . Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7 - VC with either Heh1 - VN or Heh2 - VN . Cell borders are outlined . Scale bar is 5 μm .

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A. Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7-VC with either Heh1-VN or Heh2-VN. Cell borders are outlined. Scale bar is 5 μm.


B . Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1 - VN and Snf7 - VC with either Chm7 - mCherry or Mps3 - mCherry and merge of both channels . Scale bar is 5 μm .

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B. Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1-VN and Snf7-VC with either Chm7-mCherry or Mps3-mCherry and merge of both channels. Scale bar is 5 μm.


C . Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains . Cell borders outlined . Scale bar is 5 μm .

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C. Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains. Cell borders outlined. Scale bar is 5 μm.


D , E . Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds . Data are from three independent replicates where > 225 cells per strain per replicate were quantified . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 .

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D,E. Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds. Data are from three independent replicates where > 225 cells per strain per replicate were quantified. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.


F . Deconvolved inverted fluorescence micrographs of Heh1 - VN Snf7 - VC BiFC signal in a chm7Δ strain with either an empty plasmid ( " - " ; pRS416 ) or one expressing CHM7 ( pRS416 - HA - CHM7 ) . Cell borders outlined . Scale bar is 5 μm . G . Plot of the percentage of cells with BiFC signal from F . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * * * , p ≤ 0 . 001 .

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F. Deconvolved inverted fluorescence micrographs of Heh1-VN Snf7-VC BiFC signal in a chm7Δ strain with either an empty plasmid ("-"; pRS416) or one expressing CHM7 (pRS416-HA-CHM7). Cell borders outlined. Scale bar is 5 μm.G. Plot of the percentage of cells with BiFC signal from F. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. ***, p ≤ 0.001.


A . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in the indicated strains . Scale bar is 5 μm . The percentage of cells ± SD with Chm7 - GFP foci is indicated below each panel .

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A. Deconvolved inverted fluorescence micrographs of Chm7-GFP in the indicated strains. Scale bar is 5 μm. The percentage of cells ± SD with Chm7-GFP foci is indicated below each panel.


B . Deconvolved fluorescence images of SINC - containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7 - GFP and Nup170 - mCherry ( green , red and merged images are shown ) . Scale bar is 1 μm .

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B. Deconvolved fluorescence images of SINC-containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7-GFP and Nup170-mCherry (green, red and merged images are shown). Scale bar is 1 μm.


C . Plot of the proportion of nup clusters with Chm7 - GFP enrichment in the indicated genetic backgrounds . Error bars are SD of the mean from 3 independent replicates .

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C. Plot of the proportion of nup clusters with Chm7-GFP enrichment in the indicated genetic backgrounds. Error bars are SD of the mean from 3 independent replicates.


D . Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7 - GFP and clustered Nup170 - mCherry in a nup133Δ strain . Scale bar is 1 μm .

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D. Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7-GFP and clustered Nup170-mCherry in a nup133Δ strain. Scale bar is 1 μm.


E . Plot of the total fluorescence / integrated density ( in arbitrary units ( A . U . ) ) of Chm7 - GFP within ( and outside of ) the SINC . Error bars represent SD deviation of the mean from > 50 SINC or non - SINC accumulations pooled from 3 independent replicates . p values from un - paired student ' s T - tests with Welch ' s correction . * * * * is p ≤ 0 . 0001 .

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E. Plot of the total fluorescence/integrated density (in arbitrary units (A.U.)) of Chm7-GFP within (and outside of) the SINC. Error bars represent SD deviation of the mean from > 50 SINC or non-SINC accumulations pooled from 3 independent replicates. p values from un-paired student's T-tests with Welch's correction. **** is p ≤ 0.0001.


F . Correlation of the total fluorescence intensity / integrated density of SINC - enriched Chm7 - GFP and Nup170 - mCherry within individual SINCs . Linear regression calculated from 150 SINCs pooled from 3 independent replicates ; r is the linear correlation ( Pearson ' s ) coefficient .

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F. Correlation of the total fluorescence intensity/integrated density of SINC-enriched Chm7-GFP and Nup170-mCherry within individual SINCs. Linear regression calculated from 150 SINCs pooled from 3 independent replicates; r is the linear correlation (Pearson's) coefficient.


A . 10 - fold serial dilutions of the indicated yeast strains were grown on YPD at 23 ° C for 3 days before imaging .

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A.10-fold serial dilutions of the indicated yeast strains were grown on YPD at 23°C for 3 days before imaging.


C . Deconvolved fluorescence micrographs of Sna3 - mCherry in the indicated genetic backgrounds . Scale bar is 5 μm .

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C. Deconvolved fluorescence micrographs of Sna3-mCherry in the indicated genetic backgrounds. Scale bar is 5 μm.


D . Deconvolved fluorescence micrographs ( maximum intensity projections of a z - series of images ) of GFP - Nup49 in the indicated yeast strains ( arrows indicate SINCs ) . E . Plot of proportion of cells in the indicated strains with SINCs . Error bars are the SD from the mean from 3 independent replicates quantifying > 300 cells for each strain . p values from un - paired student ' s T - tests where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

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D. Deconvolved fluorescence micrographs (maximum intensity projections of a z-series of images) of GFP-Nup49 in the indicated yeast strains (arrows indicate SINCs).E. Plot of proportion of cells in the indicated strains with SINCs. Error bars are the SD from the mean from 3 independent replicates quantifying >300 cells for each strain. p values from un-paired student's T-tests where ns is p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


A . Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7 - GFP ( percentages ± SD of the proportion of cells with Chm7 - GFP foci is at bottom of each panel , see Figure EV5B ) . Scale bar is 5 μm .

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A. Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7-GFP (percentages ± SD of the proportion of cells with Chm7-GFP foci is at bottom of each panel, see Figure EV5B). Scale bar is 5 μm.


B . Plot of the percentage of cells with the indicated number of Chm7 - GFP foci . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns represents p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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B. Plot of the percentage of cells with the indicated number of Chm7-GFP foci. Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns represents p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.


C . Deconvolved inverted fluorescencemicrographs of strains expressing Chm7 - GFP after 5 h at 23 ° C ( top ) or 37 ° C ( bottom ) . Scale bar is 5 μm . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) . D . Plots of percentage of cells from C with the indicated number of Chm7 - GFP foci at 23 ° C ( left ) and 37 ° C ( right ) . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 .

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C. Deconvolved inverted fluorescencemicrographs of strains expressing Chm7-GFP after 5 h at 23°C (top) or 37°C (bottom). Scale bar is 5 μm. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).D. Plots of percentage of cells from C with the indicated number of Chm7-GFP foci at 23°C (left) and 37°C (right). Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns is p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001.


A . Plot of the percentage of nup116Δ cells incubated for 3 h at 37 ° C with nuclear rim - like accumulations of Chm7 - GFP . Error bars are SD from the mean from 3 independent replicates of > 100 cells . p values from unpaired student ' s T - test where * * * is p ≤ 0 . 001 . B . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in nup116Δ cells at either 23 ° C or grown for 3 h at 37 ° C . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) .

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A. Plot of the percentage of nup116Δ cells incubated for 3 h at 37°C with nuclear rim-like accumulations of Chm7-GFP. Error bars are SD from the mean from 3 independent replicates of >100 cells. p values from unpaired student's T-test where *** is p ≤ 0.001.B. Deconvolved inverted fluorescence micrographs of Chm7-GFP in nup116Δ cells at either 23°C or grown for 3 h at 37°C. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).


C . Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37 ° C . N denotes nuclear interior . Scale bars are 100 nm .

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C. Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37°C. N denotes nuclear interior. Scale bars are 100 nm.


D . 10 - fold serial dilutions of indicated strains grown in medium permissive ( - URA ) or restrictive ( 5 - FOA ) of the retention of a URA3 / NUP116 plasmid . Images acquired after incubation for 3 days ( left ) or 6 days ( right ) at 23 ° C .

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D. 10-fold serial dilutions of indicated strains grown in medium permissive (-URA) or restrictive (5-FOA) of the retention of a URA3/NUP116 plasmid. Images acquired after incubation for 3 days (left) or 6 days (right) at 23°C.


A . Deconvolved fluorescence micrographs of NLS - GFP in the indicated yeast strains at either 30 ° C or after 2 h at 37 ° C . Scale bar is 5 μm . B . Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS - GFP from cells represented in A . Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain . p values from un - paired student ' s T - test where ns is p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . Only chm7Δ and chm7Δapq12Δ cells have N : C ratios below 1 . 2 ( dotted red line ) . C . Re - plotting of data from B showing the percentage of cells with a N : C NLS - GFP fluorescence ratio < 1 . 2 . Error bars are the SD from the mean from 3 independent replicates of 150 cells / strain . p values from student ' s T - test where * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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A. Deconvolved fluorescence micrographs of NLS-GFP in the indicated yeast strains at either 30°C or after 2 h at 37°C. Scale bar is 5 μm.B. Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS-GFP from cells represented in A. Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain. p values from un-paired student's T-test where ns is p > 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Only chm7Δ and chm7Δapq12Δ cells have N:C ratios below 1.2 (dotted red line).C. Re-plotting of data from B showing the percentage of cells with a N:C NLS-GFP fluorescence ratio <1.2. Error bars are the SD from the mean from 3 independent replicates of 150 cells/strain. p values from student's T-test where *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.


( B ) High magnification view of boxed regions in ( A ) pseudo - colored to show actin depletion in anaphase . Scale bar - 1 µm .

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(B) High magnification view of boxed regions in (A) pseudo-colored to show actin depletion in anaphase. Scale bar -1 µm.


( F ) Stills from time - lapse sequence of representative Hela cells expressing mNeon - Green Aurora B and Cell Mask to label cell membrane , exiting mitosis , showing the dynamic re - localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment ( arrows top ) . With knock - down of RACGAP1 , Aurora B still re - localises from the DNA to the microtubules and furrow in the midzone in early anaphase ( arrows bottom ) , but the microtubule localization is lost at later stages ( asterisk bottom ) . Scale bar - 10 µm .

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(F) Stills from time-lapse sequence of representative Hela cells expressing mNeon-Green Aurora B and Cell Mask to label cell membrane, exiting mitosis, showing the dynamic re-localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment (arrows top). With knock-down of RACGAP1, Aurora B still re-localises from the DNA to the microtubules and furrow in the midzone in early anaphase (arrows bottom), but the microtubule localization is lost at later stages (asterisk bottom). Scale bar - 10 µm.


( E ) Stills of representative Hela cells expressing Lifeact - GFP and H2B - mCherry at 10 mins following forced mitotic exit with siControl , siRACGAP1 , DMSO and 2µM ZM447439 treatment . Control siRNA , RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA , while ZM447439 treated cells fail to clear actin , quantified in ( F ) . Note , failure in actin accumulation in RACGAP1 depleted cell , asterisk . Scale bar - 10

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(E) Stills of representative Hela cells expressing Lifeact-GFP and H2B-mCherry at 10 mins following forced mitotic exit with siControl, siRACGAP1, DMSO and 2µM ZM447439 treatment. Control siRNA, RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA, while ZM447439 treated cells fail to clear actin, quantified in (F). Note, failure in actin accumulation in RACGAP1 depleted cell, asterisk. Scale bar - 10 µm


( F ) Quantification of actin intensity across the perimeter of the cells based on distance from DNA , with 5 and 6 being closest to DNA and 1 and 10 furthest away , as in cartoon ( B ) . Graph shows decrease in actin intensity closer to the DNA in siControl ( n = 26 ) , siRACGAP1 ( n = 37 ) and Control DMSO treated cells ( n = 38 cells ) , seen by levels lower than 1 ( dotted red line ) , while this decrease is absent in ZM447439 treated cells ( n = 42 cells ) . Data presented as mean ± SD .

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(F) Quantification of actin intensity across the perimeter of the cells based on distance from DNA, with 5 and 6 being closest to DNA and 1 and 10 furthest away, as in cartoon (B). Graph shows decrease in actin intensity closer to the DNA in siControl (n=26), siRACGAP1 (n=37) and Control DMSO treated cells (n=38 cells), seen by levels lower than 1 (dotted red line), while this decrease is absent in ZM447439 treated cells (n=42 cells). Data presented as mean ± SD.


( B ) Stills from time - lapse sequence of the basal most region of Hela cells expressing Lifeact - GFP and H2B - mCherry forced to undergo flat monopolar mitotic exit . While actin is cleared underneath the DNA in control DMSO treated cells , it fails to be cleared around the DNA when cells are treated with Aurora B inhibitor ZM447439 during mitotic exit . Dotted mask shows position of DNA in actin channel . Scale bar - 10µm .

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(B) Stills from time-lapse sequence of the basal most region of Hela cells expressing Lifeact-GFP and H2B-mCherry forced to undergo flat monopolar mitotic exit. While actin is cleared underneath the DNA in control DMSO treated cells, it fails to be cleared around the DNA when cells are treated with Aurora B inhibitor ZM447439 during mitotic exit. Dotted mask shows position of DNA in actin channel. Scale bar -10µm.


( C ) Quantification of clearance of actin underneath the DNA for still shown in ( B ) . While DNA under control cell starts clearing actin within 10 mins , Aurora B inhibited cells fail to do so .

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(C) Quantification of clearance of actin underneath the DNA for still shown in (B). While DNA under control cell starts clearing actin within 10 mins, Aurora B inhibited cells fail to do so.


( D ) Still images of representative Hela cells expressing Lifeact - GFP and H2B - mCherry captured at early anaphase under different conditions i . e . Control siRNA , knock - down of centralspindlin protein RACGAP1 , in the presence and absence of Aurora B kinase inhibitor ( 2µM ZM ) . Scale bar - 10 µm ( E ) Quantification of aspect ratio of cells treated with different conditions as in ( D ) at 6 mins after anaphase onset , before furrow formation . Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio , while together have a stronger impact during mitotic exit . In combined treatment , mitotic cells hardly undergo any elongation and remain spherical before re - spreading . Ordinary one - way ANOVA with multiple comparison . Each treatment is compared with siControl , * * * * p < 0 . 0001 for all comparisons . Data represented as box - whisker plot , with box showing 25th - 75th percentile values , whiskers - min to max value and line representing median . Mean ± SD for different treatments - siControl ( n = 38 ) - 1 . 455 ± 0 . 19 , ZM ( n = 32 ) - 1 . 219 ± 0 . 16 , siRACGAP1 ( n = 25 ) - 1 . 144 ± 0 . 078 and siRACGAP1 + ZM ( n = 16 ) - 1 . 013 ± 0 . 03 . Cells for different treatment pooled from n > 3 independent experiments .

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(D) Still images of representative Hela cells expressing Lifeact-GFP and H2B-mCherry captured at early anaphase under different conditions i.e. Control siRNA, knock-down of centralspindlin protein RACGAP1, in the presence and absence of Aurora B kinase inhibitor (2µM ZM). Scale bar - 10 µm (E) Quantification of aspect ratio of cells treated with different conditions as in (D) at 6 mins after anaphase onset, before furrow formation. Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio, while together have a stronger impact during mitotic exit. In combined treatment, mitotic cells hardly undergo any elongation and remain spherical before re-spreading. Ordinary one-way ANOVA with multiple comparison. Each treatment is compared with siControl, ****p<0.0001 for all comparisons. Data represented as box-whisker plot, with box showing 25th-75th percentile values, whiskers-min to max value and line representing median. Mean±SD for different treatments- siControl (n=38) - 1.455±0.19, ZM (n=32) - 1.219±0.16, siRACGAP1 (n=25) -1.144±0.078 and siRACGAP1+ZM (n=16) - 1.013±0.03. Cells for different treatment pooled from n>3 independent experiments.


( C ) Quantification of actin intensity at the poles of cells at 6 mins post anaphase onset , before furrow formation under different conditions as in ( A ) . Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin and Aurora B kinase activity are affected , while it is cleared upon RACGAP1 depletion . Data represented as mean ± SD for treatments - siControl - 0 . 98 ± 0 . 05 , siRACGAP1 - 0 . 99 ± 0 . 04 , ZM - 1 ± 0 . 039 , siRACGAP1 + ZM - 1 ± 0 . 037 . Ordinary one - way ANOVA with multiple comparison - siControl vs siRACGAP1 - n . s . , p = 0 . 896 , siControl vs ZM - * * p = 0 . 0018 , siControl vs siRACGAP1 + ZM - * * * * p < 0 . 0001 and ZM vs siRACGAP1 + ZM - p = 0 . 1528 .

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(C) Quantification of actin intensity at the poles of cells at 6 mins post anaphase onset, before furrow formation under different conditions as in (A). Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin and Aurora B kinase activity are affected, while it is cleared upon RACGAP1 depletion. Data represented as mean±SD for treatments - siControl- 0.98±0.05, siRACGAP1 - 0.99±0.04, ZM - 1±0.039, siRACGAP1+ZM - 1±0.037. Ordinary one-way ANOVA with multiple comparison- siControl vs siRACGAP1 - n.s., p=0.896, siControl vs ZM - **p=0.0018, siControl vs siRACGAP1+ZM - ****p<0.0001 and ZM vs siRACGAP1+ZM - p=0.1528.


( G ) Quantification of shortest distance between centroid of DNA and cortical actomyosin network . While chromatin is able to reach close to the cortex in both siControl and siRACGAP1 treated cells , the distance of chromatin from cortex increases upon ZM treatment and is further enhanced in the double treatment condition . Data represented as mean ± SD . siControl - 4 . 7 ± 0 . 87 , siRACGAP1 - 4 . 4 ± 0 . 64 , ZM - 5 . 47 ± 0 . 87 , siRACGAP1 + ZM - 6 . 35 ± 1 . 14 microns . Ordinary one - way ANOVA with multiple comparison , siControl vs ZM , * * * p = 0 . 002 , siControl vs siRACGAP1 , p = 0 . 2823 , siControl vs siRACGAP1 + ZM , * * * * p < 0 . 0001 , siRACGAP1 vs siRACGAP1 + ZM , * * * * p < 0 . 0001 , ZM vs siRACGAP1 , * * * * p < 0 . 0001 , ZM vs siRACGAP1 + ZM , * * * p = 0 . 0004 .

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(G) Quantification of shortest distance between centroid of DNA and cortical actomyosin network. While chromatin is able to reach close to the cortex in both siControl and siRACGAP1 treated cells, the distance of chromatin from cortex increases upon ZM treatment and is further enhanced in the double treatment condition. Data represented as mean±SD. siControl - 4.7±0.87, siRACGAP1 - 4.4±0.64, ZM - 5.47±0.87, siRACGAP1+ZM - 6.35±1.14 microns. Ordinary one-way ANOVA with multiple comparison, siControl vs ZM, ***p=0.002, siControl vs siRACGAP1, p=0.2823, siControl vs siRACGAP1+ZM, ****p<0.0001, siRACGAP1 vs siRACGAP1+ZM, ****p <0.0001, ZM vs siRACGAP1, ****p<0.0001, ZM vs siRACGAP1+ZM, ***p=0.0004 .


Differences in clinical variables including ( B ) liver fat , plasma levels of ( C ) ALT , ( D ) AST , ( E ) uric acid and ( F ) creatinine are presented in the CMA and placebo groups on Days 0 , 14 and 70 after weight loss adjustment . Adj . P indicates p value after weight loss adjustment . Statistical significance is defined based on paired Student ' s t test . p < 0 . 05 . The boxes show the distribution of the clinical parameters in different groups . The bottom and top of the boxes represent the 25th and 75th percentiles . The central band represents the median value . The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values . The sample sizes on Days 0 , 14 or 70 were marked in each boxplot .

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Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.


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Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B ) in both ( n = 4 ) ;

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Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B) in both (n=4);


A ) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups . Asterisks indicate statistical significance based on paired Student ' s t test . p < 0 . 05 ;

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A) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups. Asterisks indicate statistical significance based on paired Student's t test. p < 0.05;


B ) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine , cysteine , carnitine and nicotinamide . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM )

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B) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine, cysteine, carnitine and nicotinamide. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM)


Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM ) C ) with the 10 most significantly correlated plasma metabolites ,

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Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM) C) with the 10 most significantly correlated plasma metabolites,


Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A ) gut microbiome between CMA and placebo groups on Day 70 vs Day 0 ; B ) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups ; C ) oral microbiome between CMA and placebo groups on Day 70 vs Day 0 ; D ) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups . Each dot represents a species and it has been colored according to its corresponding phylum . Asterisks indicate statistical significance based on paired Wilcoxon signed - rank test . p < 0 . 05 . Log2FC : log2 ( fold change ) .

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Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A) gut microbiome between CMA and placebo groups on Day 70 vs Day 0; B) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups; C) oral microbiome between CMA and placebo groups on Day 70 vs Day 0; D) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups. Each dot represents a species and it has been colored according to its corresponding phylum. Asterisks indicate statistical significance based on paired Wilcoxon signed-rank test. p< 0.05. Log2FC: log2(fold change).


Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , B ) plasma level of 10 most significant inflammation related

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Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , B) plasma level of 10 most significant inflammation related proteins


Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , C ) the abundance of the species in gut

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Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , C) the abundance of the species in gut microbiome


Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with D ) the abundance of the species in oral microbiome . AST , Aspartate aminotransferase ; ALT , Alanine transaminase . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Cor . Coeff : Correlation coefficient .

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Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with D) the abundance of the species in oral microbiome. AST, Aspartate aminotransferase; ALT, Alanine transaminase. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient.


A , B ( A ) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II - S5P IF showing that SPT5 and Pol II - S5P occupied the FOS loci after serum starvation . Zoomed - in views of the merged regions are indicated by the white arrow . ( B ) Same as Fig 1A . but for FOS DNA FISH with concurrent SPT5 and NELFE IF .

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A, B (A) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II-S5P IF showing that SPT5 and Pol II-S5P occupied the FOS loci after serum starvation. Zoomed-in views of the merged regions are indicated by the white arrow. (B) Same as Fig 1A. but for FOS DNA FISH with concurrent SPT5 and NELFE IF.


E Confocal images showing that SPT5 , NELFA , Pol II - S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1 % 1 , 6 - hexanediol ( abbreviation as 1 , 6 - hex ) for 30 minutes . F Violin plot showing the number of cells containing nuclear puncta in Fig 1E . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . For SPT5 , p = 0 . 1301 . For NELFA , p = 0 . 2756 . For Pol II - S5P , p = 0 . 8179 . For AFF4 , * * * p < 0 . 0001 .

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E Confocal images showing that SPT5, NELFA, Pol II-S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1% 1,6-hexanediol (abbreviation as 1,6-hex) for 30 minutes. F Violin plot showing the number of cells containing nuclear puncta in Fig 1E. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. For SPT5, p = 0.1301. For NELFA, p = 0.2756. For Pol II-S5P, p = 0.8179. For AFF4, ***p < 0.0001. G


G - I ChIP - qPCR showing that the occupancies of Pol II ( G ) , SPT5 ( H ) , and NELFE ( I ) at the promoters and gene bodies of HSP70 , GAPDH , and MYC in HeLa cells . The HEMO gene serves as a negative control for ChIP - qPCR . Error bars represent standard error of means . Results are plotted with four technical repeats from two biological replicates . Two - tailed , unpaired Student ' s t test was performed . For Pol II ChIP , HSP70 _ promoter , * * p = 0 . 0014 ; GAPDH _ promoter , p = 0 . 8385 ; GAPDH _ gene body , * * * p < 0 . 0001 ; MYC _ promoter , p = 0 . 1765 ; MYC _ gene body , * * * p = 0 . 0002 . For SPT5 ChIP , HSP70 _ promoter , * * p = 0 . 0033 ; GAPDH _ promoter , p = 0 . 9603 ; GAPDH _ gene body , * * p = 0 . 0012 ; MYC _ promoter , p = 0 . 9898 ; MYC _ gene body , * * * p < 0 . 0001 . For NELFE ChIP , HSP70 _ promoter , p = 0 . 3913 ; GAPDH _ promoter , p = 0 . 9347 ; MYC _ promoter , p = 0 . 8558 .

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G-I ChIP-qPCR showing that the occupancies of Pol II (G), SPT5 (H), and NELFE (I) at the promoters and gene bodies of HSP70, GAPDH, and MYC in HeLa cells. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, **p = 0.0014; GAPDH_promoter, p = 0.8385; GAPDH_gene body, ***p < 0.0001; MYC_promoter, p = 0.1765; MYC_gene body, ***p = 0.0002. For SPT5 ChIP, HSP70_promoter, **p = 0.0033; GAPDH_promoter, p = 0.9603; GAPDH_gene body, **p = 0.0012; MYC_promoter, p = 0.9898; MYC_gene body, ***p < 0.0001. For NELFE ChIP, HSP70_promoter, p = 0.3913; GAPDH_promoter, p = 0.9347; MYC_promoter, p = 0.8558.


C Normalized FRAP curves for eGFP - SPT5 . The bleaching events occurred at 0 second . Confocal images were embedded within the plot showing FRAP of the in vitro eGFP - SPT5 clusters . Results shown are from three biological replicates . The arrowhead represents the bleached region .

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C Normalized FRAP curves for eGFP-SPT5. The bleaching events occurred at 0 second. Confocal images were embedded within the plot showing FRAP of the in vitro eGFP-SPT5 clusters. Results shown are from three biological replicates. The arrowhead represents the bleached region.


E Live cell imaging of HEK - 293T cells expressing eGFP - SPT5 together with mCherry - SPT5 , mCherry - SPT5ΔNTR , or mCherry - SPT5ΔCTR close to endogenous level . F Violin plot showing the clusters area ( top ) and circularity ( bottom ) of SPT5 with SPT5 , SPT5ΔNTR , or SPT5ΔCTR in Fig 2E . Each n > 20 . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . * * * p < 0 . 001 .

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E Live cell imaging of HEK-293T cells expressing eGFP-SPT5 together with mCherry-SPT5, mCherry-SPT5ΔNTR, or mCherry-SPT5ΔCTR close to endogenous level. F Violin plot showing the clusters area (top) and circularity (bottom) of SPT5 with SPT5, SPT5ΔNTR, or SPT5ΔCTR in Fig 2E. Each n > 20. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001. G


H Confocal images showing FRAP analysis of the NELFA - IDR - eGFP and SPT5 - CTR - mCherry clusters in vitro . The arrowhead represents the bleached and recovered cluster . I Normalized FRAP curves for NELFA - IDR - eGFP and SPT5 - CTR - mCherry clusters . The bleaching events occurred at 0 second . Results shown are from two biological replicates .

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H Confocal images showing FRAP analysis of the NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters in vitro. The arrowhead represents the bleached and recovered cluster. I Normalized FRAP curves for NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters. The bleaching events occurred at 0 second. Results shown are from two biological replicates.


A Western blot analysis of re - expression of mCherry - SPT5 , mCherry - SPT5ΔNTR or mCherry - SPT5ΔCTR after SPT5 depletion . α - Tubulin was used as a loading control . Results are representative of two biological replicates .

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A Western blot analysis of re-expression of mCherry-SPT5, mCherry-SPT5ΔNTR or mCherry-SPT5ΔCTR after SPT5 depletion. α-Tubulin was used as a loading control. Results are representative of two biological replicates.


B Confocal imaging of nascent RNA labeled by EU in HEK - 293T cells expressing SPT5 , SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion .

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B Confocal imaging of nascent RNA labeled by EU in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion.