A10 cells were transfected with Flag - FMRP as above and then pre - treated with rapamycin or solvent ( DMSO ) for 1 hr in serum - free media . Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β - catenin antibody . Non - programmed rabbit IgG was used as a control . Existence of Flag - FMRP in the immunocomplex was detected by Flag antibody and β - catenin by GFP

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A10 cells were transfected with Flag-FMRP as above and then pre-treated with rapamycin or solvent (DMSO) for 1 hr in serum-free media. Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β-catenin antibody. Non-programmed rabbit IgG was used as a control. Existence of Flag-FMRP in the immunocomplex was detected by Flag antibody and β-catenin by GFP antibody


A10 cells were transfected with Flag - FMRP followed by rapamycin treatment similar to ( D ) . Cells were fixed and stained for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . A loss of β - catenin signal from the perinuclear region that was seen in the control is indicated by the arrows .

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A10 cells were transfected with Flag-FMRP followed by rapamycin treatment similar to (D). Cells were fixed and stained for β-catenin (green), Flag (FMRP, red), and nucleus (blue). A loss of β-catenin signal from the perinuclear region that was seen in the control is indicated by the arrows.


( Top panel ) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were treated with 0 . 5 µM puromycin for 15 min and harvested for Western blot analysis . Total β - catenin levels are indicated , and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) was graphed . Error bars indicate standard deviation . An independent samples t - test was performed ( 2 - tailed ) , with the p - value indicated above the error bar . ( Bottom panel ) Cells were treated similarly to above and fixed and stained for β - catenin ( green ) and nucleus ( blue ) The experiment in ( A ) was repeated using A10

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(Top panel) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were treated with 0.5 µM puromycin for 15 min and harvested for Western blot analysis. Total β-catenin levels are indicated, and actin was used as a loading control. The average puromycin/actin ratio (n=3) was graphed. Error bars indicate standard deviation. An independent samples t-test was performed (2-tailed), with the p-value indicated above the error bar. (Bottom panel) Cells were treated similarly to above and fixed and stained for β-catenin (green) and nucleus (blue) The experiment in (A) was repeated using A10 cells


( i ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were harvested and subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody . The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis . Non - programed rabbit IgG was used as a control . Efficacy of Wnt - 3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β - catenin ( S33 / 37 / T41 ) by Wnt - 3a . ( ii ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . Cell lysates were then subjected to m7GTP - agarose pull - down analysis . eIF4E and tubulin were used as positive and negative controls , respectively , and phospho - β - catenin ( S33 / 37 / T41 ) Western blots were used as controls for Wnt - 3a stimulation .

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(i) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were harvested and subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody. The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis. Non-programed rabbit IgG was used as a control. Efficacy of Wnt-3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β-catenin (S33/37/T41) by Wnt-3a. (ii) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. Cell lysates were then subjected to m7GTP-agarose pull-down analysis. eIF4E and tubulin were used as positive and negative controls, respectively, and phospho-β-catenin (S33/37/T41) Western blots were used as controls for Wnt-3a stimulation.


Representative images of seed fluorescence segregation in 420 / + + in wild type ( Col ) and hcr2 . Scatterplots to the right show red ( dsRed ) and green ( eGFP ) fluorescence values in 420 / + + Col ( top ) and hcr2 ( bottom ) . Scale bars : 2 mm .

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Representative images of seed fluorescence segregation in 420/++ in wild type (Col) and hcr2. Scatterplots to the right show red (dsRed) and green (eGFP) fluorescence values in 420/++ Col (top) and hcr2 (bottom). Scale bars: 2 mm.


End - point RT - PCR analysis of HSBP in Col , hsbp - 3 , hsbp - 2 and hsbp - 1 . Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp - 3 , respectively . Image J was used to measure relative PCR band intensity for hsbp - 3 ( 53 % ) , hsbp - 2 ( 9 % ) and hsbp - 1 ( 70 % ) . TUB2 was used as an internal control . As in E , but showing immunoblot analysis of HSBP . hsbp - 3 , hsbp - 2 and hsbp - 1 accumulate about 58 % , 17 % and 77 % of HSBP levels , respectively . Coomassie - stained membrane was used as a loading control .

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End-point RT-PCR analysis of HSBP in Col, hsbp-3, hsbp-2 and hsbp-1. Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp-3, respectively. Image J was used to measure relative PCR band intensity for hsbp-3 (53%), hsbp-2 (9%) and hsbp-1 (70%). TUB2 was used as an internal control. As in E, but showing immunoblot analysis of HSBP. hsbp-3, hsbp-2 and hsbp-1 accumulate about 58%, 17% and 77% of HSBP levels, respectively. Coomassie-stained membrane was used as a loading control.


Representative images of ASY1 ( green ) and RAD51 ( red ) immunostaining in Col and hsbp - 3 . Nuclei spreads were stained with DAPI ( blue ) . Scale bars : 10 μm . Quantification of RAD51 foci numbers per cell in Col ( blue ) and hsbp - 3 ( red ) . n = 20 cells of biological replicates .

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Representative images of ASY1 (green) and RAD51 (red) immunostaining in Col and hsbp-3. Nuclei spreads were stained with DAPI (blue). Scale bars: 10 μm. Quantification of RAD51 foci numbers per cell in Col (blue) and hsbp-3 (red). n = 20 cells of biological replicates.


Representative images of MLH1 ( red ) immunostaining in Col , hsbp - 3 , hsbp - 2 , Col / Ler , and meiMIGS - HSBP Col / Ler . Nuclear DNA was stained with DAPI . Scale bar : 5 μm . Quantification of the number of MLH1 foci per cell shown in ( G ) . n ≥ 32 cells of biological replicates .

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Representative images of MLH1 (red) immunostaining in Col, hsbp-3, hsbp-2, Col/Ler, and meiMIGS-HSBP Col/Ler. Nuclear DNA was stained with DAPI. Scale bar: 5 μm. Quantification of the number of MLH1 foci per cell shown in (G). n ≥ 32 cells of biological replicates.


Representative images of HEI10 ( red ) and ASY1 ( green ) immunostaining in Col and hsbp - 3 . Scale bar : 2 μm .

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Representative images of HEI10 (red) and ASY1 (green) immunostaining in Col and hsbp-3. Scale bar: 2 μm.


Quantification of the number of HEI10 foci per cell shown in ( I ) . n ≥ 21 cells of biological replicates .

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Quantification of the number of HEI10 foci per cell shown in (I). n ≥ 21 cells of biological replicates.


Immunoblot analysis of HEI10 and HEI10 - Myc in Col , hsbp - 3 , HEI10 - myc and HEI10 - myc plants . Experiments were performed at least three times .

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Immunoblot analysis of HEI10 and HEI10-Myc in Col, hsbp-3, HEI10-myc and HEI10-myc plants. Experiments were performed at least three times.


( A - D ) The serial dilutions of post - second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2 - Line19F - FFL virions . The serum samples were serially diluted , and the detection of antibodies were measured by optical density ( OD ) at 490 nm . End - point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control ( PBS immunized animal ) .

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(A-D) The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies were measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal).


( A ) Lung RSV titers ( PFU / gram of lung tissue ) were determined in individual lungs ( n = 5 mice per group ) collected at 5 days post - RSV infection . Results were determined and presented as box and whisker plots , where boxes extends from 25th to 75th percentile , whiskers show minimum to maximum value and central band represents the median value for the group .

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(A) Lung RSV titers (PFU/gram of lung tissue) were determined in individual lungs (n = 5 mice per group) collected at 5 days post-RSV infection. Results were determined and presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.


( A ) Serum samples collected from individual mouse ( M 1 - 5 ) immunized with F 110 - 136 peptide were tested for antibody binding against F - p27 ( 110 - 136 ) peptide or F 1 - 34 peptide ( control ) in ELISA .

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(A) Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110-136) peptide or F 1-34 peptide (control) in ELISA.


( B ) A549 cells were infected with RSV ( MOI = 0 . 1 ) for 16 h , and fixed . Cells were treated with mock control rabbit sera ( left panels ) , or rabbit anti - sera against F ( center panels ) , or against F - p27 ( 110 - 136 ) peptide ( right panels ) , followed by Alexa 594 conjugated anti - Rabbit IgG ( red ) . Nuclei are stained with DAPI ( blue ) . Scale bar = 10 µm . The number of cells positive for RSV - F ( middle panel ) and RSV - p27 ( right panel ) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the ' merge ' panel . The experiments were performed twice and variation between the two independent expeiremnts was < 6 % .

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(B) A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit anti-sera against F (center panels), or against F-p27 (110-136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice and variation between the two independent expeiremnts was <6%.


Mice lungs were collected from PBS - ( mock ) vaccinated BALB / c mice at day 5 post - RSV infection , fixed , paraffin embedded , and slides were processed for imaging . Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera ( left panels ) or rabbit anti - sera against RSV F protein ( center panels ) , or against RSV F - p27 ( 110 - 136 ) peptide ( right panels ) . Scale bar = 100 µm . The percentage of cells positive for RSV - F ( middle panel ) and RSV - p27 ( right panel ) covering 500 x 500 µm of lung tissue for each mice for each antibody staining are shown . The experiments were performed twice and variation between the two independent expeiremnts was < 9 % .

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Mice lungs were collected from PBS-(mock) vaccinated BALB/c mice at day 5 post-RSV infection, fixed, paraffin embedded, and slides were processed for imaging. Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera (left panels) or rabbit anti-sera against RSV F protein (center panels), or against RSV F-p27 (110-136) peptide (right panels). Scale bar = 100 µm. The percentage of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) covering 500 x 500 µm of lung tissue for each mice for each antibody staining are shown. The experiments were performed twice and variation between the two independent expeiremnts was <9%.


( A ) Lung tissues from individual mice ( n = 5 ) at day 5 post - RSV challenge ( or uninfected control ) were used to evaluate CD4 / CD8 T cells separately for airway and distal lungs . Lung slides were fixed and stained with DAPI to visualize nuclei ( blue ) and either anti - CD4 ( green ) or anti - CD8 ( red ) T cells . The number of CD4 / CD8 T cells were determined in each lung airway and distal region . Individual CD4 + / CD8 + T cells data are presented as number of positive cells per 174 x 174 µm of lung tissue for each mice . Results are presented as box and whisker plots , where boxes extends from 25th to 75th percentile , whiskers show minimum to maximum value and central band represents the median value for the group .

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(A) Lung tissues from individual mice (n=5) at day 5 post-RSV challenge (or uninfected control) were used to evaluate CD4/CD8 T cells separately for airway and distal lungs. Lung slides were fixed and stained with DAPI to visualize nuclei (blue) and either anti-CD4 (green) or anti-CD8 (red) T cells. The number of CD4/CD8 T cells were determined in each lung airway and distal region. Individual CD4+/CD8+ T cells data are presented as number of positive cells per 174 x 174 µm of lung tissue for each mice. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.


A Western blot analysis of YTHDC1 in HEK293T and MCF - 7 knockdown samples .

A Western blot analysis of YTHDC1 in HEK293T and MCF-7 knockdown samples.


B Volcano plot of genes with significant switching to lengthened ( red ) or shortened ( blue ) 3 ' UTRs according to test of linear trend alternative to independence in HEK293T ( left panel ) and MCF - 7 cells ( right panel ) . Embedded bar plots show the number of significant genes . The X axis shows the Pearson correlation r from the linear trend alternative test , and the y axis shows the FDR .

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B Volcano plot of genes with significant switching to lengthened (red) or shortened (blue) 3' UTRs according to test of linear trend alternative to independence in HEK293T (left panel) and MCF-7 cells (right panel). Embedded bar plots show the number of significant genes. The X axis shows the Pearson correlation r from the linear trend alternative test, and the y axis shows the FDR.


E The qRT - PCR validation of APA switching . Eight genes with lengthened and shortened 3 ' UTRs in HEK293T and in MCF - 7 cells were chosen . PRPF4B , CTNNBIP1 , PHB and RCL1 were selected from HEK293T cells , and rest genes are MCF - 7 cells . KD ( Extended / Common ) / Ctr ( Extended / Common ) < 1 indicates a shorter 3 ' UTR in the knockdown sample , and vice versa . Data are presented as mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , the p values were obtained from unpaired two - tailed Student ' s t - test .

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E The qRT-PCR validation of APA switching. Eight genes with lengthened and shortened 3' UTRs in HEK293T and in MCF-7 cells were chosen. PRPF4B, CTNNBIP1, PHB and RCL1 were selected from HEK293T cells, and rest genes are MCF-7 cells. KD (Extended/Common)/Ctr (Extended/Common) <1 indicates a shorter 3' UTR in the knockdown sample, and vice versa. Data are presented as mean ± SEM of three biological replicates. *p<0.05, **p<0.01, the p values were obtained from unpaired two-tailed Student's t-test.


A Higher proportion of target genes in the list of genes with shortened 3 ' UTRs after knockdown of YTHDC1 . The p values were obtained by Fisher ' s exact test . * p = 1 . 227 × 10 - 3 , * * p = 3 . 725 × 10 - 4 , NA : no significance .

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A Higher proportion of target genes in the list of genes with shortened 3'UTRs after knockdown of YTHDC1. The p values were obtained by Fisher's exact test. *p=1.227×10-3, **p=3.725×10-4, NA: no significance.


C Peak density distribution of YTHDC1 binding sites near different poly ( A ) sites . Genes with shortened 3 ' UTR shows a higher density of YTHDC1 binding peaks near proximal APA sites compared to background genes . The density of YTHDC1 binding site peak were calculated as the number of peak in a 10 - nt interval divided by the total number of mRNAs that contained this position .

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C Peak density distribution of YTHDC1 binding sites near different poly(A) sites. Genes with shortened 3' UTR shows a higher density of YTHDC1 binding peaks near proximal APA sites compared to background genes. The density of YTHDC1 binding site peak were calculated as the number of peak in a 10-nt interval divided by the total number of mRNAs that contained this position.


E Scatter plot of protein and mRNA ratios of dual luciferase report assay with λN - BoxB system . λN , YTHDC1 and λN - YTHDC1 were co - transfected with a bicistronic vector . Two batches of experiments with three biological repeats were performed . Both luciferase activity and mRNA levels were measured . To test the effect of YTHDC1 binding on the proximal APA site , a linear model with the co - transfected genes and batch as independent variables was fitted . Data are presented as mean ± SEM of three biological replicates . * * p < 0 . 01 with t - test from linear model .

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E Scatter plot of protein and mRNA ratios of dual luciferase report assay with λN-BoxB system. λN, YTHDC1 and λN-YTHDC1 were co-transfected with a bicistronic vector. Two batches of experiments with three biological repeats were performed. Both luciferase activity and mRNA levels were measured. To test the effect of YTHDC1 binding on the proximal APA site, a linear model with the co-transfected genes and batch as independent variables was fitted. Data are presented as mean ± SEM of three biological replicates. ** p<0.01 with t-test from linear model.


F Overexpression of YTHDC1 - WT and YTHDC1 - Mut ( W377A , W428A ) revealed by Western blot .

F Overexpression of YTHDC1-WT and YTHDC1-Mut (W377A, W428A) revealed by Western blot.


G Scatter plot of qRT - PCR for APA validation . Mutation of the m6A binding site of YTHDC1 loses its inhibitory effect on proximal APA sites . Extended / Common ratios of ten genes were obtained by qRT - PCR in HEK293T cells overexpressing YTHDC1 - WT and YTHDC1 - Mut . The ratios were normalized to those of control cells . Data are presented as mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , ns : no significance . the p values were obtained from unpaired two - tailed Student ' s t - test .

G Scatter plot of qRT-PCR for APA validation. Mutation of the m6A binding site of YTHDC1 loses its inhibitory effect on proximal APA sites. Extended/Common ratios of ten genes were obtained by qRT-PCR in HEK293T cells overexpressing YTHDC1-WT and YTHDC1-Mut. The ratios were normalized to those of control cells. Data are presented as mean ± SEM of three biological replicates. *p<0.05, **p<0.01, ns: no significance. the p values were obtained from unpaired two-tailed Student's t-test.


A - C The Co - IP results of endogenous WTAP , METTL3 and METTL14 with YTHDC1 . The endogenous WTAP and YTHDC1 can be pulled down reciprocally by each other in the presence of RNase A .

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A-C The Co-IP results of endogenous WTAP, METTL3 and METTL14 with YTHDC1. The endogenous WTAP and YTHDC1 can be pulled down reciprocally by each other in the presence of RNase A.


D The Co - IP results of Myc - CBLL1 , Myc - RBM15 , Myc - ZC3H13 and Myc - VIRMA with Endogenous YTHDC1 . None of them can pull down Endogenous YTHDC1 in the presence of RNase A .

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D The Co-IP results of Myc-CBLL1, Myc-RBM15, Myc-ZC3H13 and Myc-VIRMA with Endogenous YTHDC1. None of them can pull down Endogenous YTHDC1 in the presence of RNase A.


E . Endogenous YTHDC1 interacts with RPB3 in a RNA independent way .

E. Endogenous YTHDC1 interacts with RPB3 in a RNA independent way.


A Co - IP assay using Myc - tagged 3 ' end processing factors to pull down endogenous YTHDC1 without the addition of RNase A . FIP1L1 , PABPC1 and PABPC4 can pull down endogenous YTHDC1 .

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A Co-IP assay using Myc-tagged 3' end processing factors to pull down endogenous YTHDC1 without the addition of RNase A. FIP1L1, PABPC1 and PABPC4 can pull down endogenous YTHDC1.


B Co - IP assay using Myc - FIP1L1 , Myc - PABPC1 and Myc - PABPC4 to pull down endogenous YTHDC1 in the presence of RNase A .

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B Co-IP assay using Myc-FIP1L1, Myc-PABPC1 and Myc-PABPC4 to pull down endogenous YTHDC1 in the presence of RNase A.


C Co - IP assay with antibodies against endogenous YTHDC1 and FIP1L1 .

C Co-IP assay with antibodies against endogenous YTHDC1 and FIP1L1.


D Coomassie brilliant blue staining of purified different epitope - tagged YTHDC1 and FIP1L1 .

D Coomassie brilliant blue staining of purified different epitope-tagged YTHDC1 and FIP1L1.


E An in vitro pull - down assay using purified FLAG - YTHDC1 and Myc - FIP1L1 showed that FLAG - YTHDC1 directly interacts with Myc - FIP1L1 .

E An in vitro pull-down assay using purified FLAG-YTHDC1 and Myc-FIP1L1 showed that FLAG-YTHDC1 directly interacts with Myc-FIP1L1.


F Endogenous YTHDC1 and FIP1L1 were stained with antibodies labeled with Alexa Fluor 488 and Alexa Fluor 568 fluorophores , respectively . Immunofluorescence results show that YTHDC1 and FIP1L1 have a high spatial association in the nucleus . Scale bars 15µm .

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F Endogenous YTHDC1 and FIP1L1 were stained with antibodies labeled with Alexa Fluor 488 and Alexa Fluor 568 fluorophores, respectively. Immunofluorescence results show that YTHDC1 and FIP1L1 have a high spatial association in the nucleus. Scale bars 15µm.


G Scan graph analysis and nuclear staining density quantification of YTHDC1 and FIP1L1 , indicating high colocalization in the nucleus . H Forty cells were randomly selected , and colocalization was assessed by calculating the average overlap coefficient , Pearson ' s correlation and colocalization rate according to Leica TCS SP8 microscope software . Data are presented as mean ± SD of 40 biological replicates .

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G Scan graph analysis and nuclear staining density quantification of YTHDC1 and FIP1L1, indicating high colocalization in the nucleus. H Forty cells were randomly selected, and colocalization was assessed by calculating the average overlap coefficient, Pearson's correlation and colocalization rate according to Leica TCS SP8 microscope software. Data are presented as mean ± SD of 40 biological replicates.


B Co - IP assays using truncated FIP1L1 proteins revealed that the proline - rich domain of FIP1L1 plays an important role in interacting with YTHDC1 .

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B Co-IP assays using truncated FIP1L1 proteins revealed that the proline-rich domain of FIP1L1 plays an important role in interacting with YTHDC1.


C Mutation of FIP1L1 nearly abrogates the interaction between YTHDC1 and FIP1L1 compared with wild type .

C Mutation of FIP1L1 nearly abrogates the interaction between YTHDC1 and FIP1L1 compared with wild type.


D - G Co - IP assays using different domains of YTHDC1 revealed that the N - terminus ( 1 - 344 aa ) and YTH domain ( 364 - 507 aa ) of YTHDC1 interact with FIP1L1 .

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D-G Co-IP assays using different domains of YTHDC1 revealed that the N-terminus (1-344 aa) and YTH domain (364-507 aa) of YTHDC1 interact with FIP1L1.


B YTHDC1 inhibits the interaction between Myc - FIP1L1 and FLAG - CPSF4 . A substantially suppressed level of interaction between Myc - FIP1L1 and FLAG - CPSF4 was observed when FLAG - YTHDC1 input was increased .

B YTHDC1 inhibits the interaction between Myc-FIP1L1 and FLAG-CPSF4. A substantially suppressed level of interaction between Myc-FIP1L1 and FLAG-CPSF4 was observed when FLAG-YTHDC1 input was increased.


C Knockdown of YTHDC1 enhances endogenous FIP1L1 recruitment to CPSF4 , indicating that YTHDC1 plays an important role in interfering with the 3 ' end processing complex interaction .

C Knockdown of YTHDC1 enhances endogenous FIP1L1 recruitment to CPSF4, indicating that YTHDC1 plays an important role in interfering with the 3' end processing complex interaction.


D YTHDC1 has little effect on the interaction between Myc - FIP1L1 and FLAG - PAPOLA . The interaction between Myc - FIP1L1 and FLAG - PAPOLA was not significantly affected when FLAG - YTHDC1 was increased .

D YTHDC1 has little effect on the interaction between Myc-FIP1L1 and FLAG-PAPOLA. The interaction between Myc-FIP1L1 and FLAG-PAPOLA was not significantly affected when FLAG-YTHDC1 was increased.


E Mutation of proline had little effect on the interaction between FIP1L1 and CPSF4 compared with the wild type .

E Mutation of proline had little effect on the interaction between FIP1L1 and CPSF4 compared with the wild type.


F The mutation of prolines in FIP1L1 abrogated the inhibitory effect of YTHDC1 on the interaction between FIP1L1 and CPSF4 .

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F The mutation of prolines in FIP1L1 abrogated the inhibitory effect of YTHDC1 on the interaction between FIP1L1 and CPSF4.


G qRT - PCR validation of APA site switching in a bicistronic dual luciferase system . FIP1L1 - Mut could significantly increase the ratios of Rluc / Fluc compared to FIP1L1 - WT , indicating that YTHDC1 inhibits the use of proximal APA sites by interacting with the proline - rich domain of FIP1L1 . Data are presented as mean ± SEM of three biological replicates . * * p = 8 . 77 × 10 - 4 , the p values were obtained with unpaired two - tailed Student ' s t - test .

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G qRT-PCR validation of APA site switching in a bicistronic dual luciferase system. FIP1L1-Mut could significantly increase the ratios of Rluc/Fluc compared to FIP1L1-WT, indicating that YTHDC1 inhibits the use of proximal APA sites by interacting with the proline-rich domain of FIP1L1. Data are presented as mean ± SEM of three biological replicates. **p=8.77×10-4, the p values were obtained with unpaired two-tailed Student's t-test.


B Microscopy images of fluorescence recovery following partial photobleaching of GFP - FIP1L1 . Normal solutions containing 2 . 1 mg / ml GFP - FIP1L1 were examined with a microscope at different time points . Scale bars : upper 3 µm , lower 2 . 98 µm .

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B Microscopy images of fluorescence recovery following partial photobleaching of GFP-FIP1L1. Normal solutions containing 2.1 mg/ml GFP-FIP1L1 were examined with a microscope at different time points. Scale bars: upper 3 µm, lower 2.98 µm.


C FRAP assay of GFP - FIP1L1 . Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 8 individual GFP - FIP1L1 droplets . Data are presented as mean ± SD of 8 biological replicates .

C FRAP assay of GFP-FIP1L1. Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 8 individual GFP-FIP1L1 droplets. Data are presented as mean ± SD of 8 biological replicates.


D The GFP - FIP1L1 phase separation assay was performed in normal buffer , high salt buffer and normal buffer with the addition of RNA . All these results indicated that the GFP - FIP1L1 protein displayed dynamic and liquid - like properties . Scale bars 15µm .

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D The GFP-FIP1L1 phase separation assay was performed in normal buffer, high salt buffer and normal buffer with the addition of RNA. All these results indicated that the GFP-FIP1L1 protein displayed dynamic and liquid-like properties. Scale bars 15µm.


E Sanger sequencing was used to genotype eGFP - FIP1L1 and mScarlet - YTHDC1 homozygous cell lines .

E Sanger sequencing was used to genotype eGFP-FIP1L1 and mScarlet-YTHDC1 homozygous cell lines.


F In vivo FRAP assays for eGFP - FIP1L1 and DsRed - YTHDC1 were examined by time - lapse phase - contrast imaging . The arrows represent droplet of GFP - FIP1L1 . Nine cell - independent experiments were selected for FRAP assays . Scale bars 10 µm . G , Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 15 individual GFP - FIP1L1 droplets in figure G respectively . Data are presented as mean ± SD of 15 biological replicates .

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F In vivo FRAP assays for eGFP-FIP1L1 and DsRed-YTHDC1 were examined by time-lapse phase-contrast imaging. The arrows represent droplet of GFP-FIP1L1.Nine cell-independent experiments were selected for FRAP assays. Scale bars 10 µm. G, Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 15 individual GFP-FIP1L1 droplets in figure G respectively. Data are presented as mean ± SD of 15 biological replicates.


H Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 15 individual DsRed - YTHDC1 in figure H , respectively . Data are presented as mean ± SD of 15 biological replicates .

H Quantification of average relative fluorescence intensity and its initial rate of area of photobleaching across 15 individual DsRed-YTHDC1 in figure H, respectively. Data are presented as mean ± SD of 15 biological replicates.


I Genome - edited eGFP - FIP1L1 HEK293T cells expressing Dsred - YTHDC1 - WT and Dsred - YTHDC1 - Mut were examined by confocal microscopy . DsRed - YTHDC1 - WT forms LLPS to compartmentalize FIL1L1 , but DsRed - YTHDC1 - Mut diffuses in the nucleus without droplet structure . Droplet structures are indicated by arrows . Scale bars : upper 8 . 09 µm , lower 6 . 64 µm . J Colocalization of YTHDC1 - WT and YTHDC1 - Mut in figure I was assessed by calculating the average overlap coefficient according to Leica TCS SP8 microscope software . Data are presented as mean ± SEM of 15 cells . * * * * p = 6 . 46 × 10 - 4 , the p values were obtained with unpaired two - tailed Student ' s t - test .

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I Genome-edited eGFP-FIP1L1 HEK293T cells expressing Dsred-YTHDC1-WT and Dsred-YTHDC1-Mut were examined by confocal microscopy. DsRed-YTHDC1-WT forms LLPS to compartmentalize FIL1L1, but DsRed-YTHDC1-Mut diffuses in the nucleus without droplet structure. Droplet structures are indicated by arrows. Scale bars: upper 8.09 µm, lower 6.64 µm. J Colocalization of YTHDC1-WT and YTHDC1-Mut in figure I was assessed by calculating the average overlap coefficient according to Leica TCS SP8 microscope software. Data are presented as mean ± SEM of 15 cells. ****p=6.46×10-4, the p values were obtained with unpaired two-tailed Student's t-test.


B RT - PCR analysis of RNA from mid - temporal autopsy brain sections obtained from NCI , MCI or AD subjects . Representative gel of results is shown . C Quantitation of RT - PCR products of all samples analyzed from post - mortem brain tissues ( mean s . e . m . ) . An omnibus significant effect was obtained using the one - way analysis of variance ( one - way ANOVA ) with Tukey post - test to determine the p - value . Error bars are s . e . m .

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B RT-PCR analysis of RNA from mid-temporal autopsy brain sections obtained from NCI, MCI or AD subjects. Representative gel of results is shown.C Quantitation of RT-PCR products of all samples analyzed from post-mortem brain tissues (mean s.e.m.). An omnibus significant effect was obtained using the one-way analysis of variance (one-way ANOVA) with Tukey post-test to determine the p-value. Error bars are s.e.m.


D RT - PCR analysis of RNA from the hippocampus of 1 - 2 and 3 - 7 month old non - transgenic ( WT ) or TgCRND8 ( AD ) mice . E Quantification of the relative ratio of exon 19 inclusion for TgCRND8 ( AD ) and WT mice ( mean s . e . m . ; one - way ANOVA with Tukey post - test ) . The number of samples analyzed ( n ) is indicated .

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D RT-PCR analysis of RNA from the hippocampus of 1-2 and 3-7 month old non-transgenic (WT) or TgCRND8 (AD) mice.E Quantification of the relative ratio of exon 19 inclusion for TgCRND8 (AD) and WT mice (mean s.e.m.; one-way ANOVA with Tukey post-test). The number of samples analyzed (n) is indicated.


B Top , diagram of ASOs tested below , mapped to their position of complementarity on ApoER2 . Bottom , RT - PCR of RNA isolated from HeLa cells transfected with the indicated ASO at a final concentration of 80 nM . RNA spliced forms are labeled . Quantification of the percentage inclusion is shown below the image and calculated as : Inclusion ( % ) = [ exon 19 included / ( exon 19 included + exon 19 skipped ) ] x 100 .

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B Top, diagram of ASOs tested below, mapped to their position of complementarity on ApoER2. Bottom, RT-PCR of RNA isolated from HeLa cells transfected with the indicated ASO at a final concentration of 80 nM. RNA spliced forms are labeled. Quantification of the percentage inclusion is shown below the image and calculated as: Inclusion (%)=[exon 19 included/(exon 19 included+exon 19 skipped)] x 100.


D Top , RT - PCR analysis of ApoER2 exon 19 splicing in HeLa cells depleted of SRSF1 using an SRSF1 - specific siRNA . Control is a non - specific siRNA . Bottom , Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . E Quantification of exon 19 splicing in ( D ) . Error bars show s . e . m . P - value was determined using Student ' s two - tailed t - test .

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D Top, RT-PCR analysis of ApoER2 exon 19 splicing in HeLa cells depleted of SRSF1 using an SRSF1-specific siRNA. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.E Quantification of exon 19 splicing in (D). Error bars show s.e.m. P-value was determined using Student's two-tailed t-test.


F Top , RT - PCR analysis of exon 19 splicing in a primary mouse kidney cell line depleted of SRSF1 by RNAi . Control is a non - specific siRNA . Bottom , Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . G Quantification of exon 19 splicing in E . Error bars show s . e . m . P - value was calculated using Student ' s two - tailed t - test .

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F Top, RT-PCR analysis of exon 19 splicing in a primary mouse kidney cell line depleted of SRSF1 by RNAi. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.G Quantification of exon 19 splicing in E. Error bars show s.e.m. P-value was calculated using Student's two-tailed t-test.


A Top , ASOs mapped onto the mouse ApoER2 exon 19 gene region . Bottom , RT - PCR of RNA isolated from mouse cells transfected with different ASOs . Quantification of percent exon 19 inclusion is shown below the gel image .

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A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image.


B ASO improves exon 19 inclusion in a dose - responsive manner . RT - PCR analysis of RNA from mouse cells transfected with increasing doses of ASO - 15 . The percent inclusion of exon 19 is shown below the gel image . The half - maximal effective concentration ( EC50 ) was calculated using GraphPad Prism version 6 . 0 ( GraphPad Software , San Diego , CA ) after fitting the data using nonlinear regression with normalized response and variable slope .

B ASO improves exon 19 inclusion in a dose-responsive manner. RT-PCR analysis of RNA from mouse cells transfected with increasing doses of ASO-15. The percent inclusion of exon 19 is shown below the gel image. The half-maximal effective concentration (EC50) was calculated using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA) after fitting the data using nonlinear regression with normalized response and variable slope.


D RT - PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs . C refers to a non - specific control ASO . E Quantitation of ApoER2 exon 19 inclusion from D ( mean s . e . m . , one - way ANOVA with Bonferroni multiple comparison test .

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D RT-PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs. C refers to a non-specific control ASO.E Quantitation of ApoER2 exon 19 inclusion from D (mean s.e.m., one-way ANOVA with Bonferroni multiple comparison test.


A RT - PCR analysis of ApoER2 exon 19 splicing in RNA isolated from hippocampus of 4 month old AD and WT mice treated with ASO - 21 or ASO - C at P1 - 2 . B Quantitation of exon 19 splicing . ( mean s . e . m . ; Kruskal - Wallis test with Dunn ' s multiple comparison test .

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A RT-PCR analysis of ApoER2 exon 19 splicing in RNA isolated from hippocampus of 4 month old AD and WT mice treated with ASO-21 or ASO-C at P1-2.B Quantitation of exon 19 splicing. (mean s.e.m.; Kruskal-Wallis test with Dunn's multiple comparison test.


C Immunoblot for exon 19 containing ApoER2 ( top ) and total ApoER2 ( bottom ) . β - actin was used as loading control . D Quantification of ApoER2 protein isoforms shown in C ( mean s . e . m . , student ' s t - test ) .

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C Immunoblot for exon 19 containing ApoER2 (top) and total ApoER2 (bottom). β-actin was used as loading control.D Quantification of ApoER2 protein isoforms shown in C (mean s.e.m., student's t-test).


A Input - output curves show hippocampal CA1 field excitatory postsynaptic potential ( fEPSP ) response amplitudes as a function of stimulus intensity delivered to Schaffer collateral afferent fibers . Two - way repeated - measures ANOVA revealed a significant effect of treatment ( F ( 2 , 119 ) = 31 . 62 , p < 0 . 0001 ) with the Bonferroni multiple comparisons test showing significant differences between ASO - C treated AD and age - matched ASO - C treated WT ( * p < 0 . 05 ) . Responses from ASO - 21 treated AD mice were not significantly different from ASO - C treated WT or AD mice .

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A Input-output curves show hippocampal CA1 field excitatory postsynaptic potential (fEPSP) response amplitudes as a function of stimulus intensity delivered to Schaffer collateral afferent fibers. Two-way repeated-measures ANOVA revealed a significant effect of treatment (F(2,119) = 31.62, p < 0.0001) with the Bonferroni multiple comparisons test showing significant differences between ASO-C treated AD and age-matched ASO-C treated WT (* p < 0.05). Responses from ASO-21 treated AD mice were not significantly different from ASO-C treated WT or AD mice.


B Representative paired - pulse facilitation ( PPF ) responses from ASO - C treated WT ( top ) , ASO - C treated AD ( middle ) and ASO - 21 treated AD ( bottom ) mice are shown to the left . Scale bars = 0 . 2 mV by 10 msec apply to all traces . Bar graph ( right ) summarizes the paired - pulse ratios . PPF was not significantly different in AD compared to WT samples , nor was it affected by ASO - 21 treatment .

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B Representative paired-pulse facilitation (PPF) responses from ASO-C treated WT (top), ASO-C treated AD (middle) and ASO-21 treated AD (bottom) mice are shown to the left. Scale bars = 0.2 mV by 10 msec apply to all traces. Bar graph (right) summarizes the paired-pulse ratios. PPF was not significantly different in AD compared to WT samples, nor was it affected by ASO-21 treatment.


C LTP was unaffected after ASO - 21 treatment and similar in AD compared to WT mice . To combine data from multiple slices , individually measured fEPSP rise slopes were normalized to the average baseline value taken 0 - 10 minutes before tetanic stimulation ( arrows ) . The normalized values are plotted as a function of time for ASO - C treated WT ( gray , left ) , ASO - C treated AD ( red , middle ) and ASO - 21 treated AD mice ( blue , right ) . Representative traces taken before and 50 - 60 minutes after tetanic stimulation are shown in the insets ( scale bars = 0 . 2 mV by 10 msec for all traces ) . D Graph summarizes LTP results as the ratio of mean fEPSP rise slope measured 50 - 60 minutes after tetanic stimulation divided by that taken 0 - 10 minutes before tetanic stimulation . There was no significant difference among groups . All data are shown as mean ± s . e . m and are from the same hippocampal brain slices ( n = 6 slices from 4 , 3 , and 4 WT / ASO - C , AD / ASO - C and AD / ASO - 21 , animals per group , respectively ) .

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C LTP was unaffected after ASO-21 treatment and similar in AD compared to WT mice. To combine data from multiple slices, individually measured fEPSP rise slopes were normalized to the average baseline value taken 0-10 minutes before tetanic stimulation (arrows). The normalized values are plotted as a function of time for ASO-C treated WT (gray, left), ASO-C treated AD (red, middle) and ASO-21 treated AD mice (blue, right). Representative traces taken before and 50-60 minutes after tetanic stimulation are shown in the insets (scale bars = 0.2 mV by 10 msec for all traces).D Graph summarizes LTP results as the ratio of mean fEPSP rise slope measured 50-60 minutes after tetanic stimulation divided by that taken 0-10 minutes before tetanic stimulation. There was no significant difference among groups. All data are shown as mean ± s.e.m and are from the same hippocampal brain slices (n=6 slices from 4, 3, and 4 WT/ASO-C, AD/ASO-C and AD/ASO-21, animals per group, respectively).


A Representative water - maze pathway traces of the last four trials of day 3 . Trial 1 is shown in red , trial 2 in green , trial 3 in blue and trial 4 in black . B Morris water maze ( MWM ) analysis of acquisition performance of male ( left ) and female ( right ) mice represented as the mean distance ( cm ) that mice traveled to find the submerged platform plotted per day ( mean s . e . m . ) Symbols represent statistically significant difference within the same genotype ( * AD ASO - C vs AD ASO - 21 and # WT ASO - C vs WT ASO - 21 ) or between groups with same treatment ( ΔAD ASO - C vs WT ASO - C and ^ AD ASO - 21 vs WT ASO - 21 ) on the corresponding day ( two - way repeated - measures ANOVA with Tukey multiple comparisons test ; * * p < 0 . 01 , ^ p < 0 . 05 , ΔΔΔΔp < 0 . 0001 ) . See source data for statistical results . C Morris water maze analysis integrated distance ( Zhang et al , 2014 ) ( area under the curve , AUC ) for male ( left ) and female ( right ) WT and AD mice treated with control ASO ( ASO - C ) or ASO - 21 . ( mean s . e . m . ; one - way ANOVA , Tukey post - test . D , E Correlation analysis of performance on day 3 of the MWM with exon 19 skipping among all groups of mice ( D ) or between AD mice treated with ASO - C or ASO - 21 ( E ) . Male and female mice are indicated . Pearson correlation coefficient ( r ) , covariance ( σ ) and two - tailed p - value are shown .

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A Representative water-maze pathway traces of the last four trials of day 3. Trial 1 is shown in red, trial 2 in green, trial 3 in blue and trial 4 in black.B Morris water maze (MWM) analysis of acquisition performance of male (left) and female (right) mice represented as the mean distance (cm) that mice traveled to find the submerged platform plotted per day (mean s.e.m.) Symbols represent statistically significant difference within the same genotype (*AD ASO-C vs AD ASO-21 and #WT ASO-C vs WT ASO-21) or between groups with same treatment (ΔAD ASO-C vs WT ASO-C and ^AD ASO-21 vs WT ASO-21) on the corresponding day (two-way repeated-measures ANOVA with Tukey multiple comparisons test; **p<0.01, ^p<0.05, ΔΔΔΔp<0.0001). See source data for statistical results.C Morris water maze analysis integrated distance (Zhang et al, 2014) (area under the curve, AUC) for male (left) and female (right) WT and AD mice treated with control ASO (ASO-C) or ASO-21. (mean s.e.m.; one-way ANOVA, Tukey post-test.D,E Correlation analysis of performance on day 3 of the MWM with exon 19 skipping among all groups of mice (D) or between AD mice treated with ASO-C or ASO-21 (E). Male and female mice are indicated. Pearson correlation coefficient (r), covariance (σ) and two-tailed p-value are shown.


( A ) Representative clones screened for CD24 expression under regulation of tetracycline . The cells were exposed to 1 µg / ml tetracycline for 48 h . 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti - CD24 SWA11 mAb . The membrane was reprobed with anti - tubulin antibody to assess the uniformity of sample loading .

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(A) Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 µg/ml tetracycline for 48 h. 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti-CD24 SWA11 mAb. The membrane was reprobed with anti-tubulin antibody to assess the uniformity of sample loading.


( D ) Western blot analysis of EXO - CD24 exosomes . Tetracycline ( Tet ) was used to induce CD24 expression in the cell line from which exosomes are derived . HSP70 was used as an exosomal marker .

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(D) Western blot analysis of EXO-CD24 exosomes. Tetracycline (Tet) was used to induce CD24 expression in the cell line from which exosomes are derived. HSP70 was used as an exosomal marker.


( C ) Western blot analysis of EXO - mCD24 exosomes . HSP70 was used as an exosomal marker .

(C) Western blot analysis of EXO-mCD24 exosomes. HSP70 was used as an exosomal marker.


( B ) respiratory rate and ( C ) Blood oxygen ( SpO2 ) and before treatment ( Day 1 , measured before administration of first dose ) and after treatment ( Day 7 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for Blood oxygen ( SpO2 ) and respiratory rate . these are presented graphically via box plots before and after treatment .

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(B) respiratory rate and (C) Blood oxygen (SpO2) and before treatment (Day 1, measured before administration of first dose) and after treatment (Day 7). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for Blood oxygen (SpO2) and respiratory rate. these are presented graphically via box plots before and after treatment.


Systemic CRP levels . Values are normalized against baseline value ( Day 0 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for CRP levels . these are presented graphically via box plots before and after treatment ( n = 35 patients )

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Systemic CRP levels. Values are normalized against baseline value (Day 0). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for CRP levels. these are presented graphically via box plots before and after treatment (n=35 patients)


( B ) Systemic cytokine levels . Cytokine levels were measured during ( Day 3 / 4 ) or after ( Day 7 / 8 ) treatment with EXO - CD24 using Quantibody Multiplex ELISA Array . Values are normalized against baseline value ( Day 0 ) . Samples from 11 patients were analysed . Results from 8 patients are shown ( results for 3 patients are not shown because one or more baseline results were below detection level ) . Box plot of Systemic cytokine levels by days after treatment ( minimum , maximum , median , first quartile , third quartile ) .

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(B) Systemic cytokine levels. Cytokine levels were measured during (Day 3/4) or after (Day 7/8) treatment with EXO-CD24 using Quantibody Multiplex ELISA Array. Values are normalized against baseline value (Day 0). Samples from 11 patients were analysed. Results from 8 patients are shown (results for 3 patients are not shown because one or more baseline results were below detection level). Box plot of Systemic cytokine levels by days after treatment (minimum, maximum, median, first quartile, third quartile).


( C ) During the course of EXO - CD24 treatment and follow - up , cytokine levels were measured for the rest of the patients ( n = 24 patients ) .

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(C) During the course of EXO-CD24 treatment and follow-up, cytokine levels were measured for the rest of the patients (n=24 patients).


( D ) Monitoring systemic IL - 6 cytokine level following EXO - CD24 treatment . Samples from 17 patients , representing the 4 experimental groups , were analysed using the BD ™ Cytometric Bead Array ( CBA ) .

(D) Monitoring systemic IL-6 cytokine level following EXO-CD24 treatment. Samples from 17 patients, representing the 4 experimental groups, were analysed using the BD™ Cytometric Bead Array (CBA).


RNA was extracted from young fertile adults fed ad libitum or fasted for 6 h . ddCts ( delta delta cycle thresholds ) were calculated normalizing to ama - 1 and the efficiency of the primer sets as previously described . Means + s . e . m . are depicted . lipl - 1 to lipl - 5 , n = 6 independent experiments . lipl - 6 to lipl - 8 , n = 3independent experiments . Significant t - test derived P - values are indicated . WT , wild type .

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RNA was extracted from young fertile adults fed ad libitum or fasted for 6 h. ddCts (delta delta cycle thresholds) were calculated normalizing to ama-1 and the efficiency of the primer sets as previously described. Means+s.e.m. are depicted. lipl-1 to lipl-5, n = 6 independent experiments. lipl-6 to lipl-8, n = 3independent experiments. Significant t-test derived P-values are indicated. WT, wild type.


( a ) Representative confocal images of LIPL - 1 : : GFP and LIPL - 3 : : TagRFP localization relative to the lysosomal marker PGP - 2 show that LIPL - 1 and LIPL - 3 are localized to the lysosomal - related organelle .

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(a) Representative confocal images of LIPL-1::GFP and LIPL-3::TagRFP localization relative to the lysosomal marker PGP-2 show that LIPL-1 and LIPL-3 are localized to the lysosomal-related organelle.


( b ) Acid lipase activity , measured in 1 - day adult whole lysates , shows that lipl - 1lipl - 3 double mutant animals have reduced acidic lipolytic capacity . Mean ± s . d . are presented relative to wild type ( WT ) ; significant differences are indicated ; n = 4 independent experiments .

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(b) Acid lipase activity, measured in 1-day adult whole lysates, shows that lipl-1lipl-3 double mutant animals have reduced acidic lipolytic capacity. Mean±s.d. are presented relative to wild type (WT); significant differences are indicated; n = 4 independent experiments.


( c ) Oil Red O ( ORO ) staining and fatty acid methyl ester ( FAME ) analyses of wild - type and lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant L3 larvae show that lysosomal lipases regulate cytosolic fat stores ( see Fig . 2d for adult measurements ) . Means ± s . e . m . are presented relative to wild type ; significant differences are indicated ; n = 3 independent experiments . ( d ) ORO quantification of wild - type and lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant young adults fasted for 4 h shows that LIPL - 1 and LIPL - 3 contribute to fat mobilization following fasting . ( Mean percentage ± s . e . m . relative to fed wild type ) . Wild - type fasted worms show 20 % less ORO signal than animals fed ad libitum ( P ≤ 0 . 001 ) , whereas lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant worms show 7 % reduction in ORO signal ( a difference that is not significant to well - fed fat levels at P ≤ 0 . 01 but it is significant at P ≤ 0 . 05 ) . n = 3 independent experiments .

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(c) Oil Red O (ORO) staining and fatty acid methyl ester (FAME) analyses of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant L3 larvae show that lysosomal lipases regulate cytosolic fat stores (see Fig. 2d for adult measurements). Means±s.e.m. are presented relative to wild type; significant differences are indicated; n = 3 independent experiments. (d) ORO quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults fasted for 4 h shows that LIPL-1 and LIPL-3 contribute to fat mobilization following fasting. (Mean percentage ± s.e.m. relative to fed wild type). Wild-type fasted worms show 20% less ORO signal than animals fed ad libitum (P≤0.001), whereas lipl-1(tm1954) lipl-3(tm4498) double mutant worms show 7% reduction in ORO signal (a difference that is not significant to well-fed fat levels at P≤0.01 but it is significant at P≤0.05). n = 3 independent experiments.


( e ) Representative transmission electron microscopy images of well - fed and 6 h - fasted wild - type and lipl - 1lipl - 3 mutant animals ( 11 , 500 × ) show that the lipl mutants have more lipid - droplet stores in ad libitum fed and fasted conditions . Quantification of vesicle number and size is depicted as mean + s . e . m . , n = 4 independent experiments .

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(e) Representative transmission electron microscopy images of well-fed and 6 h-fasted wild-type and lipl-1lipl-3 mutant animals (11,500×) show that the lipl mutants have more lipid-droplet stores in ad libitum fed and fasted conditions. Quantification of vesicle number and size is depicted as mean+s.e.m., n = 4 independent experiments.


( f ) Representative images of well - fed transgenic animals expressing LIPL - 3 : : TagRFP and ATGL : : GFP shows that LIPL - 3 does not localize to lipid droplets .

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(f) Representative images of well-fed transgenic animals expressing LIPL-3::TagRFP and ATGL::GFP shows that LIPL-3 does not localize to lipid droplets.


( g ) ORO staining and quantification of wild - type and lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant young adults treated post - developmentally ( from L4 ) with RNAi against the essential autophagy genes lgg - 1 and lgg - 2 or vector control show that lipl - 1 lipl - 3 and the autophagy genes lgg - 1 lgg - 2 are in the same fat regulatory pathway . ( Mean percentage + s . e . m . relative to wild type on vector control is depicted . ) n = 4 independent experiments .

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(g) ORO staining and quantification of wild-type and lipl-1(tm1954) lipl-3(tm4498) double mutant young adults treated post-developmentally (from L4) with RNAi against the essential autophagy genes lgg-1 and lgg-2 or vector control show that lipl-1 lipl-3 and the autophagy genes lgg-1 lgg-2 are in the same fat regulatory pathway. (Mean percentage+s.e.m. relative to wild type on vector control is depicted.) n = 4 independent experiments.


( a ) Expression of lipl genes in well - fed mxl - 3 ( ok1947 ) and mxl - 3 ( tm2580 ) young adults normalized to same age well - fed wild - type ( WT ) worms shows that mxl - 3 loss of function is sufficient to induce lipl - 1 to 3 and lipl - 5 . Mean ddCts + s . e . m . are depicted . n = 6 independent experiments for lipl - 1 to lipl - 5 , and n = 4 independent experiments for lipl - 6 to lipl - 8 .

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(a) Expression of lipl genes in well-fed mxl-3(ok1947) and mxl-3(tm2580) young adults normalized to same age well-fed wild-type (WT) worms shows that mxl-3 loss of function is sufficient to induce lipl-1 to 3 and lipl-5. Mean ddCts+s.e.m. are depicted. n = 6 independent experiments for lipl-1 to lipl-5, and n = 4 independent experiments for lipl-6 to lipl-8.


( b ) L4 larvae were fasted and RNA was extracted at the indicated times . Mean ddCts + s . e . m . show that the response mxl - 3 orchestrates is transient . Time 0 , n = 6 independent experiments ; time 3 - 12 h , n = 4 independent experiments ; time 18 - 24 h , n = 2 independent experiments .

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(b) L4 larvae were fasted and RNA was extracted at the indicated times. Mean ddCts+s.e.m. show that the response mxl-3 orchestrates is transient. Time 0, n = 6 independent experiments; time 3-12 h, n = 4 independent experiments; time 18-24 h, n = 2 independent experiments.


( c ) Immunostainings of well - fed or 6 or 12 h fasted MXL - 3 : : GFP young adults are presented . Quantification of GFP - positive nuclei relative to total intestinal nuclei ( 4 , 6 - diamidino - 2 - phenylindole , DAPI ) of two independent experiments shows that MXL - 3 transiently delocalizes from the intestinal nuclei during fasting .

(c) Immunostainings of well-fed or 6 or 12 h fasted MXL-3::GFP young adults are presented. Quantification of GFP-positive nuclei relative to total intestinal nuclei (4,6-diamidino-2-phenylindole, DAPI) of two independent experiments shows that MXL-3 transiently delocalizes from the intestinal nuclei during fasting.


( d ) ChIP - quantitative PCR ( ChIP - qPCR ) analysis of well - fed and 6 h fasted mixed - stage worms expressing MXL - 3 : : GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region ( CACTAT site − 88 of ama - 1 gene ) shows that MXL - 3 vacates the lipl promoters during early fasting . Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl - 1 and lipl - 3 genes were used . A representative experiment is presented ; see raw data of two independent experiments in Supplementary Table S9 .

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(d) ChIP-quantitative PCR (ChIP-qPCR) analysis of well-fed and 6 h fasted mixed-stage worms expressing MXL-3::GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region (CACTAT site −88 of ama-1 gene) shows that MXL-3 vacates the lipl promoters during early fasting. Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl-1 and lipl-3 genes were used. A representative experiment is presented; see raw data of two independent experiments in Supplementary Table S9.


( e ) Wild - type or lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl - 3 RNAi plates , incubated for 12 h at 20 ° C , and processed for total FAMEs ; as a reference , an aliquot of wild - type young adults on control RNAi bacteria was fasted for 6 h . FAME quantification ( mean percentage of fed wild type ± s . e . m . ) shows that acute mxl - 3 inactivation , as fasting , reduces fat stores in a lipl - dependent manner . n = 3 independent experiments . NSD , P - value > 0 . 05 .

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(e) Wild-type or lipl-1(tm1954) lipl-3(tm4498) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl-3 RNAi plates, incubated for 12 h at 20 °C, and processed for total FAMEs; as a reference, an aliquot of wild-type young adults on control RNAi bacteria was fasted for 6 h. FAME quantification (mean percentage of fed wild type ±s.e.m.) shows that acute mxl-3 inactivation, as fasting, reduces fat stores in a lipl-dependent manner. n = 3 independent experiments. NSD, P-value>0.05.


( a ) Transcriptional levels of hlh - 30 and the mxl - 3 - dependent lipl genes measured in 5 h fasted wild - type ( WT ) or hlh - 30 ( tm1978 ) young - adult worms depicted as mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms show that hlh - 30 is induced following fasting and HLH - 30 induces lysosomal lipolysis . N = 4 independent experiments for lipl data , n = 2 independent experiments for hlh - 30 mRNA . HLH - 30 deficiency fully abrogates lipl - 2 , 3 and 5 ( P0 . 0001 ) , and impairs lipl - 1 ( P0 . 01 ) transcriptional activation following fasting .

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(a) Transcriptional levels of hlh-30 and the mxl-3-dependent lipl genes measured in 5 h fasted wild-type (WT) or hlh-30(tm1978) young-adult worms depicted as mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms show that hlh-30 is induced following fasting and HLH-30 induces lysosomal lipolysis. N = 4 independent experiments for lipl data, n = 2 independent experiments for hlh-30 mRNA. HLH-30 deficiency fully abrogates lipl-2, 3 and 5 (P0.0001), and impairs lipl-1 (P0.01) transcriptional activation following fasting.


( b ) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults . Mean + s . e . m . of three independent experiments shows that inhibition of TOR is sufficient to induce hlh - 30 transcription .

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(b) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults. Mean+s.e.m. of three independent experiments shows that inhibition of TOR is sufficient to induce hlh-30 transcription.


( c ) L3 animals carrying the rescuing construct hlh - 30P : : HLH - 30 : : mGFP : : hlh - 30 3 ′ UTR ( mGFP , monomeric GFP , and UTR , untranslated region ) , well fed or fasted for 8 h show that HLH - 30 is enriched in intestinal nuclei of fasted worms ( exposure time : well fed , 500 ms ; fasted , 100 ms ) .

(c) L3 animals carrying the rescuing construct hlh-30P::HLH-30::mGFP::hlh-30 3′UTR (mGFP, monomeric GFP, and UTR, untranslated region), well fed or fasted for 8 h show that HLH-30 is enriched in intestinal nuclei of fasted worms (exposure time: well fed, 500 ms; fasted, 100 ms).


( d ) ORO staining of wild - type or hlh - 30 ( tm1978 ) young adults fasted for 8 h reveals that hlh - 30 is required for optimal lipid mobilization on fasting . Mean percentages + s . e . m . are shown below relative to well - fed worms treated in parallel , n = 3 independent experiments .

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(d) ORO staining of wild-type or hlh-30(tm1978) young adults fasted for 8 h reveals that hlh-30 is required for optimal lipid mobilization on fasting. Mean percentages+s.e.m. are shown below relative to well-fed worms treated in parallel, n = 3 independent experiments.


( e ) mxl - 3 and hlh - 30 transcriptional levels in wild - type , hlh - 30 ( tm1978 ) and mxl - 3 ( ok1947 ) mutants in the basal and fasted states show that mxl - 3 transcription is hlh - 30 independent , and hlh - 30 transcription is mxl - 3 independent . Expression is presented relative to wild - type fed animals as mean ddCt + s . e . m . No significant differences relative to wild type in the same feeding state were observed , n = 3 independent experiments .

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(e) mxl-3 and hlh-30 transcriptional levels in wild-type, hlh-30(tm1978) and mxl-3(ok1947) mutants in the basal and fasted states show that mxl-3 transcription is hlh-30 independent, and hlh-30 transcription is mxl-3 independent. Expression is presented relative to wild-type fed animals as mean ddCt+s.e.m. No significant differences relative to wild type in the same feeding state were observed, n = 3 independent experiments.


( f ) Representative ChIP - qPCR analysis of well - fed and 6 h fasted mixed stage worms expressing HLH - 30 : : GFP presented as in Fig . 3d shows that HLH - 30 occupies the lipl promoters during early fasting . See raw data of two independent experiments in Supplementary Table S9 .

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(f) Representative ChIP-qPCR analysis of well-fed and 6 h fasted mixed stage worms expressing HLH-30::GFP presented as in Fig. 3d shows that HLH-30 occupies the lipl promoters during early fasting. See raw data of two independent experiments in Supplementary Table S9.


( g ) Transcriptional levels of the mxl - 3 - dependent lipl genes in wild - type , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant young - adult worms show that hlh - 30 suppresses the constitutive - induction - of - the - lipl - genes phenotype of mxl - 3 mutant animals ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent experiments .

(g) Transcriptional levels of the mxl-3-dependent lipl genes in wild-type, mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant young-adult worms show that hlh-30 suppresses the constitutive-induction-of-the-lipl-genes phenotype of mxl-3 mutant animals (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments.


( a ) LRO ( lysosome - related organelle ) live Nile red - stained images of hlh - 30 ( tm1978 ) and wild - type ( WT ) L3 larvae starved for 60 h show that hlh - 30 is required for the expansion of the lysosomal compartment following fasting . Quantification as mean ± s . e . m . relative to fasted wild - type worms is also presented ; n = 3 independent experiments .

(a) LRO (lysosome-related organelle) live Nile red-stained images of hlh-30(tm1978) and wild-type (WT) L3 larvae starved for 60 h show that hlh-30 is required for the expansion of the lysosomal compartment following fasting. Quantification as mean±s.e.m. relative to fasted wild-type worms is also presented; n = 3 independent experiments.


( b ) Transcriptional levels of the autophagy genes lgg - 1 , lgg - 2 and atg - 16 . 2 in fasted wild - type and hlh - 30 ( tm1978 ) young - adult worms show that transcriptional activation of autophagy following fasting is hlh - 30 dependent ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent

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(b) Transcriptional levels of the autophagy genes lgg-1, lgg-2 and atg-16.2 in fasted wild-type and hlh-30(tm1978) young-adult worms show that transcriptional activation of autophagy following fasting is hlh-30 dependent (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments


( c ) The mean percentage ( + s . d . ) of L1 larvae alive after 48 h fasting in minimum media shows that hlh - 30 ( tm1978 ) mutant animals are sensitive to starvation . n = 3 independent experiments . For L4 survival see Supplementary Fig . S6g .

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(c) The mean percentage (+ s.d.) of L1 larvae alive after 48 h fasting in minimum media shows that hlh-30 (tm1978) mutant animals are sensitive to starvation. n = 3 independent experiments. For L4 survival see Supplementary Fig. S6g.


( a ) Expression analyses of the liver of C57BL / 6J 9 - week females fasted overnight ( ON ) for 10 h relative to siblings feeding ad libitum show that LipA ( mouse lysosomal acid lipase ) , Map1lc3a ( mammalian lgg - 1 / lgg - 2 ) and Tfeb ( mammalian hlh - 30 ) but not Max ( mammalian mxl - 3 ) are transcriptionally linked to nutrients in the mouse liver ( median + s . e . m . fold change ; ddCt ) . Levels of expression were normalized to ActB and Cog2 as internal controls ; all but Max differences are significant ( P0 . 05 ) , n = 3 independent experiments . Cyp4a14 is a positive control .

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(a) Expression analyses of the liver of C57BL/6J 9-week females fasted overnight (ON) for 10 h relative to siblings feeding ad libitum show that LipA (mouse lysosomal acid lipase), Map1lc3a (mammalian lgg-1/lgg-2) and Tfeb (mammalian hlh-30) but not Max (mammalian mxl-3) are transcriptionally linked to nutrients in the mouse liver (median+s.e.m. fold change; ddCt). Levels of expression were normalized to ActB and Cog2 as internal controls; all but Max differences are significant (P0.05), n = 3 independent experiments. Cyp4a14 is a positive control.


( b ) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2 , 4 or 8 h , compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media , show that LAL ( human lysosomal acid lipase ) , MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes , and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent ( median dCt + s . e . m . ) . Levels of expression were normalized to ACTB as internal control ; all butMAX differences are significant ( P0 . 05 ) , n = 3 independent experiments . IGFBP is a positive control . EBSS , Earles ' s balanced salt solution .

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(b) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2, 4 or 8 h, compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media, show that LAL (human lysosomal acid lipase), MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes, and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent (median dCt+s.e.m.). Levels of expression were normalized to ACTB as internal control; all butMAX differences are significant (P0.05), n = 3 independent experiments. IGFBP is a positive control. EBSS, Earles's balanced salt solution.


( a ) Wild - type ( WT ) , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant were used in longevity epistasis analyses , revealing that hlh - 30 suppresses the mxl - 3 extended lifespan phenotype . Animals were incubated at 20 ° C and transferred every other day until cessation of reproduction . The cumulative survival curve is depicted . Kaplan - Meier statistics , calculated using SSPS 17 , are also shown .

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(a) Wild-type (WT), mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant were used in longevity epistasis analyses, revealing that hlh-30 suppresses the mxl-3 extended lifespan phenotype. Animals were incubated at 20°C and transferred every other day until cessation of reproduction. The cumulative survival curve is depicted. Kaplan-Meier statistics, calculated using SSPS 17, are also shown.


( b ) Live Nile red quantification of wild - type and lipl - 1lipl - 3 double mutant worms shows that lipl - 1 and lipl - 3 contribute to the clearance of lipids from the endocytic pathway . Mean + s . e . m . signal intensity is shown as percentage relative to wild type ; significant differences are indicated ; n = 4 independent experiments .

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(b) Live Nile red quantification of wild-type and lipl-1lipl-3 double mutant worms shows that lipl-1 and lipl-3 contribute to the clearance of lipids from the endocytic pathway. Mean+s.e.m. signal intensity is shown as percentage relative to wild type; significant differences are indicated; n = 4 independent experiments.


( c , d ) Animals overexpressing LIPL - 1 or LIPL - 3 were incubated at 25 ° C and transferred every other day until cessation of reproduction . Data presented as in Fig . 7a show that activated lysosomal lipolysis extends C . elegans lifespan . a Survival presented as mean lifespan ( LS ) ± s . e . m . b Number of uncensored animals ( animals that crawled off the plate , bagged , exploded or became contaminated were censored ) .

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(c,d) Animals overexpressing LIPL-1 or LIPL-3 were incubated at 25 °C and transferred every other day until cessation of reproduction. Data presented as in Fig. 7a show that activated lysosomal lipolysis extends C. elegans lifespan. a Survival presented as mean lifespan (LS) ± s.e.m. b Number of uncensored animals (animals that crawled off the plate, bagged, exploded or became contaminated were censored).


( A ) Top 2 significantly overrepresented expression patterns for ERS UP and ERS DOWN genes following PHx . Data show changes in expression at 4 , 10 , 48h and 1 week after PHx ( 0h ) for genes comprised within each model profile of dynamic expression identified by STEM .

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(A) Top 2 significantly overrepresented expression patterns for ERS UP and ERS DOWN genes following PHx. Data show changes in expression at 4, 10, 48h and 1 week after PHx (0h) for genes comprised within each model profile of dynamic expression identified by STEM.


( C ) Bagplots showing the breadth of transcriptomic changes for the indicated datasets . Genes were positioned based on their basal expression levels in the control conditions and their FC ( Log2 ) in the indicated ( patho ) physiological context . The dark blue area is the " bag " ( 50 % of the data points around the median , which is indicated by a red cross ) while the light blue area delimits the " loop " Red dots are outliers .

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(C) Bagplots showing the breadth of transcriptomic changes for the indicated datasets. Genes were positioned based on their basal expression levels in the control conditions and their FC (Log2) in the indicated (patho)physiological context. The dark blue area is the "bag" (50% of the data points around the median, which is indicated by a red cross) while the light blue area delimits the "loop" Red dots are outliers.


( D - E ) Box plots showing normalized expression in liver ( D ) and liver - specificity index ( E ) of LIVER - ID genes , UBQ genes and other genes . Liver - specificity index was calculated as the difference of normalized expression in liver ( 2 replicates ) and mean of normalized expression in control tissues ( 2 replicates per tissue ) using data from BioGPS and is reported as Log2 . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - way ANOVA with Welch ' s correction and Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison test was used to assess statistical significance , * P < 0 . 05 .

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(D-E) Box plots showing normalized expression in liver (D) and liver-specificity index (E) of LIVER-ID genes, UBQ genes and other genes. Liver-specificity index was calculated as the difference of normalized expression in liver (2 replicates) and mean of normalized expression in control tissues (2 replicates per tissue) using data from BioGPS and is reported as Log2. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.


( F ) Similar analyses using LIVER - ID genes .

(F) Similar analyses using LIVER-ID genes.


Box plots showing Log2 FC ERS / Control in MPH ( 3 independent experiments ) ( G ) for LIVER - ID genes , UBQ genes and other genes . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - way ANOVA with Welch ' s correction and Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison test was used to assess statistical significance , * P < 0 . 05 .

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Box plots showing Log2 FC ERS/Control in MPH (3 independent experiments) (G) for LIVER-ID genes, UBQ genes and other genes. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.


Box plots showing Log2 FC ERS / Control in mouse liver ( 3 mice per group ) ( H ) for LIVER - ID genes , UBQ genes and other genes . Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers . One - way ANOVA with Welch ' s correction and Dunnett ' s Modified Tukey - Kramer pairwise multiple comparison test was used to assess statistical significance , * P < 0 . 05 .

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Box plots showing Log2 FC ERS/Control in mouse liver (3 mice per group) (H) for LIVER-ID genes, UBQ genes and other genes. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's Modified Tukey-Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05.


( I ) Enrichment plots from gene set enrichment analyses ( GSEA ) performed using LIVER - ID genes as the gene set and transcriptomic changes induced by acute ERS in MPH ( upper panel ) or mouse liver ( lower panel ) as the ranked gene list . NES and FDR ( as in all subsequent GSEA panels ) are the normalized enrichment score and the false discovery rate provided by the GSEA software , respectively .

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(I) Enrichment plots from gene set enrichment analyses (GSEA) performed using LIVER-ID genes as the gene set and transcriptomic changes induced by acute ERS in MPH (upper panel) or mouse liver (lower panel) as the ranked gene list. NES and FDR (as in all subsequent GSEA panels) are the normalized enrichment score and the false discovery rate provided by the GSEA software, respectively.


Genes repressed by ERS in MPH ( 3 independent experiments ) ( J ) were ranked based on their Log2 FC ERS / Control and divided into quartiles ( increased repression from Q1 to Q4 ) . The fraction of LIVER - ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1 . Chi - square test with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 .

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Genes repressed by ERS in MPH (3 independent experiments) (J) were ranked based on their Log2 FC ERS/Control and divided into quartiles (increased repression from Q1 to Q4). The fraction of LIVER-ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1. Chi-square test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05.


Genes repressed by ERS in mouse liver ( 3 mice per group ) ( K ) were ranked based on their Log2 FC ERS / Control and divided into quartiles ( increased repression from Q1 to Q4 ) . The fraction of LIVER - ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1 . Chi - square test with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 .

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Genes repressed by ERS in mouse liver (3 mice per group) (K) were ranked based on their Log2 FC ERS/Control and divided into quartiles (increased repression from Q1 to Q4). The fraction of LIVER-ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1. Chi-square test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05.


RT - qPCR analyses of selected ERS UP and LIVER - ID genes monitoring expression changes induced by acute ERS in MPH ( 3 to 9 independent experiments ) ( L ) The bar graphs show means ± SD ( standard deviations ) . One - sample t - test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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RT-qPCR analyses of selected ERS UP and LIVER-ID genes monitoring expression changes induced by acute ERS in MPH (3 to 9 independent experiments) (L) The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


RT - qPCR analyses of selected ERS UP and LIVER - ID genes monitoring expression changes induced by acute ERS in mouse liver ( 5 to 7 mice per group ) ( M ) . The bar graphs show means ± SD ( standard deviations ) . One - sample t - test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS / Control is statistically different from 0 , * P < 0 . 05 .

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RT-qPCR analyses of selected ERS UP and LIVER-ID genes monitoring expression changes induced by acute ERS in mouse liver (5 to 7 mice per group) (M). The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean Log2 FC ERS/Control is statistically different from 0, *P < 0.05.


( N ) Heatmaps showing normalized expression of ERS UP and ERS DOWN genes from MPH ( upper panel ) or mouse liver ( lower panel ) in mouse liver at the indicated stages of development .

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(N) Heatmaps showing normalized expression of ERS UP and ERS DOWN genes from MPH (upper panel) or mouse liver (lower panel) in mouse liver at the indicated stages of development.


( O ) Average expression of ERS DOWN genes from MPH in single - cells from the hepatobiliary lineage . The hepatoblast - to - hepatocyte and hepatoblast - to - cholangiocyte differentiation paths are indicated with arrows .

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(O) Average expression of ERS DOWN genes from MPH in single-cells from the hepatobiliary lineage. The hepatoblast-to-hepatocyte and hepatoblast-to-cholangiocyte differentiation paths are indicated with arrows.


( A ) Comparison of transcriptomics ( data from 3 independent experiments ) and H3K27ac ChIP - seq ( data from 3 independent experiments ) from MPH . Genes were assigned to H3K27ac regions The number of ERS DOWN genes is indicated relative to the number of ERS UP genes for the 3 categories of H3K27ac regions . Fisher ' s exact test with BH correction for multiple testing was used to assess statistical significance , * P < 0 . 05 , # P < 0 . 05 .

(A) Comparison of transcriptomics (data from 3 independent experiments) and H3K27ac ChIP-seq (data from 3 independent experiments) from MPH. Genes were assigned to H3K27ac regions The number of ERS DOWN genes is indicated relative to the number of ERS UP genes for the 3 categories of H3K27ac regions. Fisher's exact test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05, #P < 0.05.


( B ) The number of LIVER - ID and UBQ genes is indicated relative to the number of other genes for the 3 categories of H3K27ac regions . The H3K27ac ChIP - seq data were obtained from 3 independent MPH experiments . Chi - square test with BH correction for multiple testing was used to assess statistical significance , # P < 0 . 05 .

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(B) The number of LIVER-ID and UBQ genes is indicated relative to the number of other genes for the 3 categories of H3K27ac regions. The H3K27ac ChIP-seq data were obtained from 3 independent MPH experiments. Chi-square test with BH correction for multiple testing was used to assess statistical significance, #P < 0.05.


( C ) Multidimensional scaling ( MDS ) was performed and transcriptional regulator co - recruitment was depicted using density plots for regions with increased ( a ) , decreased ( b ) or unchanged ( c ) H3K27ac levels in MPH upon acute ERS . The boxed area ( d ) represents a subset of transcriptional regulators ( TR ) with a high degree of co - binding in H3K27ac DOWN regions . LIVER - ID TFs are depicted in red .

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(C) Multidimensional scaling (MDS) was performed and transcriptional regulator co-recruitment was depicted using density plots for regions with increased (a), decreased (b) or unchanged (c) H3K27ac levels in MPH upon acute ERS. The boxed area (d) represents a subset of transcriptional regulators (TR) with a high degree of co-binding in H3K27ac DOWN regions. LIVER-ID TFs are depicted in red.


( D ) Heatmaps showing the percentage of enhancers overlapping H3K27ac down regions . Enhancers were first split into those bound or not by a given LIVER - ID TF ( right ) and then based on co - binding of additional LIVER - ID TFs ( defining 3 subgroups with 0 - 2 , 3 - 5 or 6 - 8 co - bound additional LIVER - ID TFs ) .

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(D) Heatmaps showing the percentage of enhancers overlapping H3K27ac down regions. Enhancers were first split into those bound or not by a given LIVER-ID TF (right) and then based on co-binding of additional LIVER-ID TFs (defining 3 subgroups with 0-2, 3-5 or 6-8 co-bound additional LIVER-ID TFs).