Figure 6B 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 . 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 . 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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Figure 6B 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.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.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.


Figure 7B 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 . 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 . 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 . 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 .

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

Figure 7B 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.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.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.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.


Figure 1B 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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Figure 1B 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.


Figure 2B 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 . 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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Figure 2B 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.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.


Figure 3A 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 . 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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Figure 3A 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.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.


Figure 4A 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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Figure 4A 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).


Figure 5A 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 . 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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Figure 5A 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.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).


Figure 6A 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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Figure 6A 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.


Figure 1 ( 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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Figure 1(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.


Figure 4 ( C ) Western blot analysis of EXO - mCD24 exosomes . HSP70 was used as an exosomal marker .

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Figure 4(C) Western blot analysis of EXO-mCD24 exosomes. HSP70 was used as an exosomal marker.


Figure 7 ( 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 . ( D ) Representative X - ray image .

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Figure 7(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.(D) Representative X-ray image.


Figure 8Systemic 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 ) . ( 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 ) .

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

Figure 8Systemic 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).(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).


figf1RNA 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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figf1RNA 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.


figf2 ( 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 . ( 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 . ( 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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figf2(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.(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.(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.


figf3 ( 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 . ( 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 . ( 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 .

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

figf3(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.(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.(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.


figf4 ( 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 . ( 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 . ( 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 . ( 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 .

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figf4(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.(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.(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.(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.


figf5 ( 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 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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figf5(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 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.


figf6 ( 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 .

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

figf6(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.


figf7 ( 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 . ( 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 ) .

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

figf7(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.(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).


Figure 1 ( 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 . ( 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 . 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 . ( 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 . 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 . 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 . ( 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 .

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

Figure 1(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.(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.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.(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.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.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.(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.


Figure 2 ( 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 . ( 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 ) . ( E ) Enrichment plots from GSEA performed using LIVER - ID TFs 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 . ( F ) RT - qPCR analyses of selected LIVER - ID TFs monitoring expression changes induced by acute ERS in MPH ( 4 to 7 independent experiments ) . The bar graph shows 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 . ( G ) RT - qPCR analyses of selected ERS UP and LIVER - ID TF genes monitoring expression changes induced by 4h ERS in MPH pre - treated or not for 30min with 5mM PBA ( 3 independent experiments ) . Mean Log2 FC ERS / Control are shown . The bar graph shows means ± SD ( standard deviations ) . Two - way ANOVA with Bonferroni ' s post - hoc test was used to assess statistical significance , * P < 0 . 05 . ( H - I ) Nuclear extracts ( H ) and chromatin fractions ( I ) from MPH were subjected to Western blot with antibodies against HNF4A , NR1H4 / FXR , FOXA2 / HNF3B or DDIT3 / CHOP . LMNA or histone H3 were used as loading controls . Results obtained from 3 independent biological replicates are shown .

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

Figure 2(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.(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).(E) Enrichment plots from GSEA performed using LIVER-ID TFs 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.(F) RT-qPCR analyses of selected LIVER-ID TFs monitoring expression changes induced by acute ERS in MPH (4 to 7 independent experiments). The bar graph shows 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.(G) RT-qPCR analyses of selected ERS UP and LIVER-ID TF genes monitoring expression changes induced by 4h ERS in MPH pre-treated or not for 30min with 5mM PBA (3 independent experiments). Mean Log2 FC ERS/Control are shown. The bar graph shows means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.(H-I) Nuclear extracts (H) and chromatin fractions (I) from MPH were subjected to Western blot with antibodies against HNF4A, NR1H4/FXR, FOXA2/HNF3B or DDIT3/CHOP. LMNA or histone H3 were used as loading controls. Results obtained from 3 independent biological replicates are shown.


Figure 3 ( A ) RT - qPCR analyses of Hlf , Tef , Dbp and Nfil3 expression monitoring changes induced by acute ERS in MPH ( 3 to 5 independent experiments ) ( left panel ) or mouse liver ( 5 mice per group ) ( right panel ) . 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 . ( B ) Total protein extracts from MPH ( left panel ) or mouse liver ( right panel ) were subjected to Western blot with an antibody against NFIL3 . ACTB was used as loading control . ( C ) Chromatin fractions from MPH ( upper panel ) or mouse liver ( lower panel ) were subjected to Western blot with an antibody against NFIL3 . Histone H3 was used as loading control . ( D ) Enrichment scores from GSEA performed using LIVER - ID genes repressed by acute ERS in MPH ( MPH ERS DOWN ) as the gene set and liver transcriptomic changes induced by acute ERS and / or deletion of Nfil3 ( NFIL3 KO ) as the ranked gene lists were integrated and corrected for multiple testing using the BubbleGUM tool . For the NFIL3 KO ERS vs WT ERS comparison , the Core Enrichment genes ( i . e . the subset of genes that contributes most to the enrichment result ) were subjected to functional enrichment analyses using the ToppGene Suite . The top ranked KEGG Pathway with its Bonferroni - corrected p - value is shown . ( E ) Box plots showing mRNA expression for 5 genes from the Core Enrichment from D involved in xenobiotic metabolism issued from the transcriptomic analyses . Shown are Log2 FC relative to the mean normalized expression in the WT Control group ( 5 mice per group ) . 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 . Two - way ANOVA with Bonferroni ' s post - hoc test was used to assess statistical significance , * P < 0 . 05 . ( F ) The Integrated Genome Browser ( IGB ) was used to visualize ChIP - seq profiles for NFIL3 ( green ) and several LIVER - ID TFs ( red ) in the mouse liver at the Gsta3 gene locus . Levels of H3K27ac in MPH and cells from the non - parenchymal fraction ( NPC ) are shown in blue . The grey bar indicates the position of a BRD4 SE .

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

Figure 3(A) RT-qPCR analyses of Hlf, Tef, Dbp and Nfil3 expression monitoring changes induced by acute ERS in MPH (3 to 5 independent experiments) (left panel) or mouse liver (5 mice per group) (right panel). 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.(B) Total protein extracts from MPH (left panel) or mouse liver (right panel) were subjected to Western blot with an antibody against NFIL3. ACTB was used as loading control.(C) Chromatin fractions from MPH (upper panel) or mouse liver (lower panel) were subjected to Western blot with an antibody against NFIL3. Histone H3 was used as loading control.(D) Enrichment scores from GSEA performed using LIVER-ID genes repressed by acute ERS in MPH (MPH ERS DOWN) as the gene set and liver transcriptomic changes induced by acute ERS and/or deletion of Nfil3 (NFIL3 KO) as the ranked gene lists were integrated and corrected for multiple testing using the BubbleGUM tool. For the NFIL3 KO ERS vs WT ERS comparison, the Core Enrichment genes (i.e. the subset of genes that contributes most to the enrichment result) were subjected to functional enrichment analyses using the ToppGene Suite. The top ranked KEGG Pathway with its Bonferroni-corrected p-value is shown.(E) Box plots showing mRNA expression for 5 genes from the Core Enrichment from D involved in xenobiotic metabolism issued from the transcriptomic analyses. Shown are Log2 FC relative to the mean normalized expression in the WT Control group (5 mice per group). 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. Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.(F) The Integrated Genome Browser (IGB) was used to visualize ChIP-seq profiles for NFIL3 (green) and several LIVER-ID TFs (red) in the mouse liver at the Gsta3 gene locus. Levels of H3K27ac in MPH and cells from the non-parenchymal fraction (NPC) are shown in blue. The grey bar indicates the position of a BRD4 SE.


Figure 4 ( A ) BRD4 occupancy ( left panels ) and H3K27ac levels ( right panels ) at regulatory regions associated with ERS UP or LIVER - ID genes ( 8 regions at ERS gene loci and 10 regions at LIVER - ID gene loci were assessed by ChIP - qPCR in MPH ( 10 independent experiments ) or mouse liver ( 10 mice per group ) to define changes induced by acute ERS . 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 - 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 . ( B ) Left panel , BRD4 mRNA ( 4 independent experiments ) or protein expression levels ( 5 independent experiments ; densitometric quantification in MPH subjected to acute ERS . The bar graphs show means ± SD ( standard deviations ) . Student ' s t - test was used to assess statistical significance . Right panel , Total protein extracts from MPH pre - treated for 3h with 0 . 01µM MZ1 followed by addition of 1µM thapsigargin ( ERS ) for 4h were subjected to Western blot with an antibody against BRD4 . LMNA was used as loading control . ( C ) Heatmaps showing Log2 FC ( relative to the DMSO / Control condition ) for 5 ERS UP and LIVER - ID genes issued from RT - qPCR analyses of MPH pre - treated with 500nM JQ1 ( left ) or 0 . 01µM MZ1 ( right ) followed by addition of 1µM thapsigargin ( ERS ) for 4h ( 3 to 6 independent experiments ) . Two - way ANOVA with Bonferroni ' s post - hoc test was used to assess statistical significance . ( D ) Heatmaps showing average H3K27ac ChIP - seq signals in MPH at LIVER - ID domains overlapping ( + ) or not ( - ) with BRD4 SE . The arrow indicates the position of gene transcriptional start sites . ( E ) Box plots showing Log2 FC ERS / Control in MPH ( 3 independent experiments ) ( left panel ) or mouse liver ( 3 mice per group ) ( right panel ) for genes associated with LIVER - ID + BRD4 SE or LIVER - ID - BRD4 SE 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 . Student ' s t - test was used to assess statistical significance , * P < 0 . 05 .

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

Figure 4(A) BRD4 occupancy (left panels) and H3K27ac levels (right panels) at regulatory regions associated with ERS UP or LIVER-ID genes (8 regions at ERS gene loci and 10 regions at LIVER-ID gene loci were assessed by ChIP-qPCR in MPH (10 independent experiments) or mouse liver (10 mice per group) to define changes induced by acute ERS. 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-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.(B) Left panel, BRD4 mRNA (4 independent experiments) or protein expression levels (5 independent experiments; densitometric quantification in MPH subjected to acute ERS. The bar graphs show means ± SD (standard deviations). Student's t-test was used to assess statistical significance. Right panel, Total protein extracts from MPH pre-treated for 3h with 0.01µM MZ1 followed by addition of 1µM thapsigargin (ERS) for 4h were subjected to Western blot with an antibody against BRD4. LMNA was used as loading control.(C) Heatmaps showing Log2 FC (relative to the DMSO/Control condition) for 5 ERS UP and LIVER-ID genes issued from RT-qPCR analyses of MPH pre-treated with 500nM JQ1 (left) or 0.01µM MZ1 (right) followed by addition of 1µM thapsigargin (ERS) for 4h (3 to 6 independent experiments). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance.(D) Heatmaps showing average H3K27ac ChIP-seq signals in MPH at LIVER-ID domains overlapping (+) or not (-) with BRD4 SE. The arrow indicates the position of gene transcriptional start sites.(E) Box plots showing Log2 FC ERS/Control in MPH (3 independent experiments) (left panel) or mouse liver (3 mice per group) (right panel) for genes associated with LIVER-ID + BRD4 SE or LIVER-ID - BRD4 SE 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. Student's t-test was used to assess statistical significance, *P < 0.05.


Figure 5 ( A ) RT - qPCR analyses of 21 LIVER - ID genes in MPH which were treated with 1µM thapsigargin ( ERS ) for 4h , 8h , 16h or 24h ( 4 independent experiments ) . For each gene , the timepoint showing maximal repression by ERS was recorded . The heatmap depicts the percentage of genes maximally repressed by ERS at each timepoint . ( B ) RT - qPCR analyses of Hnf4a , Nr1h4 and Foxa2 expression in MPH treated with vehicle ( Control ) or 1µM thapsigargin ( ERS ) for 1h or 4h ( 4 independent experiments ) . The bar graph shows 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 . ( C ) Densitometric quantification of the protein expression data for the 1h timepoint ( average of 9 biological replicates ) and for the 4h timepoint ( average of 3 biological replicates ) . The bar graph shows 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 . ( D ) Nuclear extracts from MPH pre - treated for 30min with 1µM ISRIB followed by addition of 1µM thapsigargin ( ERS ) for 1h were subjected to Western blot with antibodies against HNF4A , NR1H4 , FOXA2 or DDIT3 . LMNA was used as loading control . Results obtained from 3 independent biological replicates are shown . ( E ) Densitometric quantification of the protein expression data shown in panel ( D ) . Repression by ERS in the ISRIB condition ( average of 3 biological replicates ) is shown relative to repression by ERS in vehicle condition . The bar graph shows means ± SD ( standard deviations ) . One - sample t - test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100 % , * P < 0 . 05 . ( F ) Nuclear extracts from MPH pre - treated for 30min with 10µM MG132 or 10µM PP2 followed by addition of 1µM thapsigargin ( ERS ) for 1h were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A , NR1H4 , FOXA2 or XBP1S . LMNA was used as loading control . Results obtained from 2 independent biological replicates are shown . ( G ) Densitometric quantification of the protein expression data shown in panel ( F ) Repression by ERS in MG132 condition ( average of 4 biological replicates ) is shown relative to repression by ERS in vehicle condition . The bar graph shows means ± SD ( standard deviations ) . One - sample t - test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100 % , * P < 0 . 05 . ( H ) ( Left ) Cytoplasmic extracts issued from the MPH ( 2 biological replicates ) were subjected to Western blot with antibodies against p - Y416 - SRC or total SRC . ACTB was used as loading control . ( Right ) Densitometric quantification of the protein expression data . The bar graphs show means ± SD ( standard deviations ) . Two - way ANOVA with Bonferroni ' s post - hoc test was used to assess statistical significance . ( I ) Densitometric quantification of the protein expression data Repression by ERS in the PP2 condition ( average of 4 biological replicates ) is shown relative to repression by ERS in vehicle condition . The bar graph shows means ± SD ( standard deviations ) . One - sample t - test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100 % . ( J ) RT - qPCR analyses of selected ERS UP and LIVER - ID TF genes monitoring expression changes induced by 4h ERS in MPH pre - treated or not for 30min with 10µM PP2 ( 3 independent experiments ) . Mean Log2 FC ERS / Control are shown . The bar graph shows means ± SD ( standard deviations ) . Two - way ANOVA with Bonferroni ' s post - hoc test was used to assess statistical significance , * P < 0 . 05 .

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

Figure 5(A) RT-qPCR analyses of 21 LIVER-ID genes in MPH which were treated with 1µM thapsigargin (ERS) for 4h, 8h, 16h or 24h (4 independent experiments). For each gene, the timepoint showing maximal repression by ERS was recorded. The heatmap depicts the percentage of genes maximally repressed by ERS at each timepoint.(B) RT-qPCR analyses of Hnf4a, Nr1h4 and Foxa2 expression in MPH treated with vehicle (Control) or 1µM thapsigargin (ERS) for 1h or 4h (4 independent experiments). The bar graph shows 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.(C) Densitometric quantification of the protein expression data for the 1h timepoint (average of 9 biological replicates) and for the 4h timepoint (average of 3 biological replicates). The bar graph shows 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.(D) Nuclear extracts from MPH pre-treated for 30min with 1µM ISRIB followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot with antibodies against HNF4A, NR1H4, FOXA2 or DDIT3. LMNA was used as loading control. Results obtained from 3 independent biological replicates are shown. (E) Densitometric quantification of the protein expression data shown in panel (D). Repression by ERS in the ISRIB condition (average of 3 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.(F) Nuclear extracts from MPH pre-treated for 30min with 10µM MG132 or 10µM PP2 followed by addition of 1µM thapsigargin (ERS) for 1h were subjected to Western blot or Simple Western immunoassay with antibodies against HNF4A, NR1H4, FOXA2 or XBP1S. LMNA was used as loading control. Results obtained from 2 independent biological replicates are shown. (G) Densitometric quantification of the protein expression data shown in panel (F) Repression by ERS in MG132 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%, *P < 0.05.(H) (Left) Cytoplasmic extracts issued from the MPH (2 biological replicates) were subjected to Western blot with antibodies against p-Y416-SRC or total SRC. ACTB was used as loading control. (Right) Densitometric quantification of the protein expression data. The bar graphs show means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance.(I) Densitometric quantification of the protein expression data Repression by ERS in the PP2 condition (average of 4 biological replicates) is shown relative to repression by ERS in vehicle condition. The bar graph shows means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine if the mean relative repression is statistically different from 100%.(J) RT-qPCR analyses of selected ERS UP and LIVER-ID TF genes monitoring expression changes induced by 4h ERS in MPH pre-treated or not for 30min with 10µM PP2 (3 independent experiments). Mean Log2 FC ERS/Control are shown. The bar graph shows means ± SD (standard deviations). Two-way ANOVA with Bonferroni's post-hoc test was used to assess statistical significance, *P < 0.05.