EMBO/soda-nel huggingface dataset:
Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly (2016)
Figure 1

Figure 1. (a) Schematic representation of U2-pre-mRNA interaction. Partial sequences/secondary structures of vertebrate and S. cerevisiae U2 are schematically shown. Also shown are the base-pairing interactions between the U2 branch site recognition region (BSRR) and the pre-mRNA branch site (BS). Pseudouridines (Ψ) are indicated. Enzymes responsible for yeast U2 pseudouridylation at the three conserved sites are also indicated. (b) Temperature-sensitive growth assay. Yeast strains (wild-type or deletion mutants, indicated on the left) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 (indicated on the top), and grown at various temperatures (indicated at the bottom). (c) Blockage of pseudouridylation by deletion of specific pseudouridylase genes. RNAs isolated from wild-type and pseudouridylase-deletion strains (indicated on the top) were used for U2 pseudouridylation assay (CMC modification followed by primer-extension). Primer-extension pauses/stops correspond to Ψ sites (indicated on the left and right). Note: The above-background signal of Ψ42 in the snr81 lane (and to some extent, in the snr81 pus7 lane) is likely caused by the presence of strong Ψ44 band. This is a primer-extension artifact, which often occurs when there is a strong Ψ signal in the neighboring position. Alternatively, it is possible that having Ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 (albeit inefficiently) (Perhaps Pus1, which is responsible for Ψ44 formation, can be a bit “processive”). Lanes 1-8 and lanes 9-16 are from two separate gels. (d) Restoration of U2 pseudouridylation by an artificial box H/ACA RNA. RNAs isolated from various yeast strains (indicated on the top), including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 (lanes 9 and 10), were assayed for pseudouridylation [see legend to (c)].
Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly (2016)
Figure 2

Figure 2. (a) Growth assay using the ACT1-CUP1 reporter system. Yeast cells carrying the ACT1-CUP1 reporter were deleted of pseudouridylase genes (indicated on the left), and were then assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom). (b) Growth rescue by restoration of pseudouridylation. The wild-type strain [from (a)] or the snr81Δ pusΔ strain [also from (a)], which exhibited the most severe growth-deficiency phenotype, was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42, position 44, or both (indicated). The resulting cells were assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom). (c) Schematic representation of U2-pre-mRNA interaction. The U2 BSRR sequence and the pre-mRNA branch site sequence are shown. The arrows indicate several point mutations at the pre-mRNA branch site. Stem-loops I, IIa and IIb are also indicated. (d) Splicing assay using the wild-type and mutant ACT1-CUP1 reporters. RNAs isolated from the wild-type and the snr81Δ pusΔ strains (indicated on the top), which carry the wild-type ACT1-CUP1 reporter pre-mRNA or any of the mutant ACT1-CUP1 reporter pre-mRNAs [indicated, also see (c)], were used for splicing assay (primer-extension analysis). The un-spliced pre-mRNA, lariat intermediate, and spliced mRNA are indicated. A primer complementary to U6 was also used (as an internal control) in the assay, and the U6 band is indicated as well. In addition, the growth phenotype of each strain (in various concentrations of [Cu2+]) is also shown. Lanes 1-12, lanes 13 and 14, and lanes 15 and 16 are from separate gels. (e) Quantification of ACT1-CUP1 mRNA levels. Spliced ACT1-CUP1 mRNA levels were calculated relative to U6 in each lane. The quantification was based on three independent experiments. (f) Relative improvement/reduction for the mutant reporters. Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency (mRNA/U6) of each mutant reporter to that of WT reporter (set at 1).
Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly (2016)
Figure 3

Figure 3. Synthetic lethality assay. The Prp5 domain structure, and the mutations in some of the domains (indicated) are schematized at the top. Each of these PRP5 genes (wild-type or mutants, plasmid-borne) was used to replace the chromosomal PRP5. The numbers in parentheses are the relative ATPase activities of the wild-type (SAT) Prp5 and some of the Prp5 mutants tested before (Xu & Query, 2007). The U2 status, either containing Ψ42 and Ψ44 (WT-U2 and snR81Δ pus1Δ with pΨ42 or Ψ44) or lacking Ψ42 and Ψ44 (snR81Δ pus1Δ), is indicated (on the right). Upon transformation with the plasmids carrying PRP5 genes (wild-type or any of the mutants), cells were grown on the regular medium (-FOA) or on medium containing 5-FOA (+FOA).
Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly (2016)
Figure 5

Figure 5. (a) Prp5's ATPase activity assay using synthetic U2. The 5' sequence of S. cerevisiae U2 is shown (top). The three Ψs (35, 42 and 44) as well as the branch site recognition sequence (underlined) are indicated. In the ATPase assay (bottom), no RNA (lane 3), or an equal amount of LiCl-precipitated RNA (lane 1) or tRNA (lane 2), a short U2 fragment (nts 1-76) (lane 4), a fully pseudouridylated short U2 fragment (nts 1-76) (lane 5), a long U2 fragment (nts 1-120) (lane 6), or a fully pseudouridylated long U2 fragment (nts 1-120) (lane 7), was used. The un-hydrolyzed ATP and hydrolyzed product ADP are indicated. Lanes 1 and 2, lanes 3-5, and lanes 6 and 7 are from separate gels. (b) Quantification of Prp5's ATPase activity activated by in vitro transcribed U2. Prp5's ATPase activity shown in (a) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (b) correspond to the lane numbers of (a). Quantification was based on three independent experiments. (c) U2-Prp5 binding assay. FLAG-Prp5 and a radiolabeled short (nts 1-76) (lanes 1 and 2) or long (nts 1-120) (lanes 5 and 6) U2 fragment, or FLAG-Prp5 and a radiolabeled fully pseudouridylated short (nts 1-76) (lanes 3 and 4) or long (nts 1-120) (lanes 7 and 8) U2 fragment, were incubated under the conditions used for ATPase activity assay. Anti-FLAG IP was then carried out. Co-precipitated (lanes 2, 4, 6 and 8) and un-precipitated (lanes 1, 3, 5 and 7) U2 RNAs were analyzed by electrophoresis. The U2 band is indicated. (d) Quantification of Prp5-U2 binding. Relative intensities of U2 bands shown in (c) were calculated [setting the un-precipitated uridine-containing U2 (lane 1 and 5) to 1]. The numbers at the bottom of (d) correspond to the lane numbers of (c). Quantification was based on three independent experiments. (e) Prp5's ATPase activity assay using cellular U2. U2 RNA isolated from the wild-type strain (lanes 2 and 4) or from the snR81Δ pus1Δ strain (lanes 6 and 8) was used for the ATPase activity assay. In odd-numbered lanes, no RNA was added. Both the wild-type Prp5 (lanes 1-4) and the mutant Prp5 (GAR) (lanes 5-8) were tested. (f) Quantification of Prp5's ATPase activity activated by U2 from cells. Prp5's ATPase activity shown in (e) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (f) correspond to the lane numbers of (e). Quantification was based on four independent experiments. (g) U2 level in the ATPase activity reactions. RNAs from lanes 2 and 4 in (c) were recovered, and U2 level was measured using primer-extension. Lanes 1-5, a titration of U2 snRNA. Lane 6, U2 from the wild-type strain [lane 2 of (c)]. Lane 7, U2 from the snR81Δ pus1Δ strain [lane 4 of (c)].
Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly (2016)
Figure 6

Figure 6. (a) Native gel analysis of pre-splicing complexes. Pre-splicing complex assembly was carried out in the test tube with Prp5-reconstituted cell extracts and labeled pre-mRNA. Lane 1, Prp5-depleted wild-type cell extract. Lane 2, Prp5-depleted wild-type cell extract, plus wild-type Prp5. Lane 3, Prp5-depleted snR81Δ pus1Δ cell extract, plus wild-type Prp5. Lane 4, Prp5-depleted wild-type cell extract, plus mutant Prp5 (GAR). Lane 5, Prp5-depleted snR81Δ pus1Δ cell extract, plus mutant Prp5 (GAR). The pre-splicing complexes A and B are indicated. (b) DMS in vivo probing. After being exposed to DMS, yeast cells were lysed, total RNA collected, and primer-extension analysis carried out (left panel). Lane 1, cells that were not exposed to DMS. Lane 2, wild-type cells exposed to DMS. Lane 3, snR81Δ pus1Δ cells exposed to DMS. Lane 4, snR81Δ pus1Δ cells, transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44, and exposed to DMS. Lane 5, snR81Δ pus1Δ cells, transformed with a plasmid carrying a control guide RNA gene with random guide sequences, and exposed to DMS. The nucleotides in parentheses are DMS-modified C and A residues that are one nucleotide after the actual primer-extension stops (indicated by the arrows). A partial U2 sequence (along with the BSL structure) is shown (right panel). DMS-modified adenosine and cytosine in the BSL are also indicated. (c) Residue accessibility quantification. Relative accessibility (relative band intensity) was quantified based on three independent experiments. The intensity of every band in each lane was normalized against the C46 band (the least changed band; compare lanes 2-5 in (b)) in its respective lane [(intensity of a band) / (intensity of C46 in the same lane)]. Each C46-normailzed band in lanes 3-5 (refer to (b)) was then further normalized against its respective counterpart in lane 2 [(the value of a C46-normalized band in lanes 3-5) / (the value of its counterpart in lane 2)]. The error bars represent the standard deviations of the measurements.
Reprogramming of glucocorticoid receptor function by hypoxia (2021)
Figure 1

Figure 1. HIFs are activated in mouse liver during hypoxia.

(A-C) The effect of hypoxia was estimated in C57BL/6J mice (n=3) by an HRE-luciferase (HRE-luc) reporter plasmid at indicated time points. All mice were injected according to body weight. (A) Experimental set-up. (B) Imaging of luciferase activity (purple signal) in the liver of control mice and mice under normoxic and hypoxic conditions at indicated time points. Red circles indicate the region of interest selected for the measurement of the luciferase activity. (C) Bioluminescent photon counts normalized to the PBS control group in liver of mice in normoxia and hypoxia at indicated time points. P-values were calculated using one-way ANOVA followed by post-hoc Tukey's test to correct for multiple testing during the pairwise multiple comparisons.

(D, E) Imaging of luciferase activity in the liver of HIF1aAlbKO (D) and HIF2aAlbKO (E) mice after a hydrodynamic tail vein injection with a HRE-luc reporter plasmid or control (PBS) according to body weight followed by 6h normoxia or hypoxia.

(F, G) Bioluminescent photon counts normalized to the PBS control group in liver of (F) HIF1aAlbKO and (G) HIF2aALbKO mice and their wild-type littermates 6h after normoxia of hypoxia.

Data information: All bars represent mean ± SEM. Each individual data point represent individual mice. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. *** P<0.001; ** P≤ 0.01; * P≤ 0.05. N = normoxia, H = hypoxia. Signal around the perimeter of the Petri dishes represent aspecific luciferase signal.
Reprogramming of glucocorticoid receptor function by hypoxia (2021)
Figure 3

Figure 3. Induction of specific DEX responsive genes in hypoxia.

Female C57BL/6J mice were put in normoxia or hypoxia for 6h or 24h, injected i.p. with PBS or DEX (10 mg/kg) and livers were isolated for genome-wide transcriptomics via RNA-seq. N=3 per group for a single RNA-seq.

(A, B) Examples of DEX-responsive genes in hypoxia 6h (A) and 24h (B) which were not induced by DEX in normoxia.

(C-E) ChIP-seq on liver derived from mice which were subjected to hypoxia (6h and 24h), followed by DEX (10 mg/kg) injection and 2h later sacrificed for liver isolation. (C) Box plots showing the LFC of genes responsive to DEX in hypoxia, but not in normoxia (6h and 24h). The central band represents the median. The box ranges from Q1 to Q3 which represents the interquartile range (IQR = Q3 - Q1) and covers the central 50% of the data. The whiskers illustrate the minimum (Q1 - 1.5*IQR) and maximum (Q3 + 1.5*IQR) of the data. Outliers are shown as dots. N=3 biological replicates per condition. (D) Histogram showing the coverage per position in a region 350 bp up- and downstream of the GR DNA-binding peaks found in hypoxia after PBS or DEX stimulation. (E) Examples of specific GR DNA-binding peaks, associated with the DEX-induced gene Slc22a5 in hypoxia (6h and 24h).

(F) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h and stimulated with DEX. Slc25a30 expression was measured in the liver via RT-qPCR. N=3 per group, one experiment.

Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ***P<0.001, **P<0.01, *P≤0.05.
Reprogramming of glucocorticoid receptor function by hypoxia (2021)
Figure 4

Figure 4. Hypoxia causes activation of GR by HPA axis stimulation.

(A, B) The effect of hypoxia on the GR response to DEX stimulation was studied via RNA-seq, n=3 per group. Venn diagram depicting the number of genes upregulated (up) or downregulated (dn) by DEX in normoxia and by hypoxia after 6h (A) or 24h (B) (LFC > 1 or LFC < -1 and P ≤ 0.05).

(C) Heatmap representing log2 values of shared stress GRE genes (counts) induced by hypoxia 6h and 24h and induced by DEX in normoxia (LFC > 1 and P ≤ 0.05).

(D-G) Female C57BL/6J and ADX mice were put in normoxia or hypoxia for 6h or 24h, n=6/group, two independent experiments. (D, E) Confirmation of RNA-seq data via RT-qPCR [6h (D) or 24h (E)]. (F, G) Plasma GC concentration [6h (F) or 24h (G)].

(H-J) Female C57BL/6J mice were put in normoxia or hypoxia for the indicated time points (2h, 6h and 24h). (H) Hypothalamic Crh mRNA expression levels were determined via RT-qPCR. (I) ACTH levels were measured in the plasma. N=3-5 per group, one experiment. P-values were analysed with one-way ANOVA. (J) Plasma GC concentration, mice in normoxia are depicted as black circles, mice in hypoxia are depicted as white squares. N=5-6 per group.

(K) Female C57BL/6J mice were put in normoxia or hypoxia for 6h and injected i.p. with 5 mg RU486 or vehicle (DMSO). Liver was isolated and stress GRE gene expression was measured via RT-qPCR. N=5 per group, one experiment.

(L) Female WT and GRdim/dim mice were put in normoxia or hypoxia for 6h and liver was isolated. Stress GRE gene expression was measured via RT-qPCR. N=5-9 per group, two independent experiments.

(M) HIF1aHIF2afl/fl and HIF1aHIF2aAlbKO mice were put in hypoxia for 6h. Stress GRE genes were measured in the liver via RT-qPCR. N=4 per group, one experiment.

(N) Expression levels of DEX responsive genes in normoxia and unique DEX responsive genes induced by hypoxia were measured in the liver of ADX mice after DEX stimulation during hypoxia (24h) via RT-qPCR. N=3-4 per group, one experiment.

Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.
Reprogramming of glucocorticoid receptor function by hypoxia (2021)
Figure 5

Figure 5: Involvement of hypothalamic HIF1α and HIF2α in activation of the HPA axis during hypoxia.

(A) Female C57BL/6J mice were put in normoxia or hypoxia for 2h, hypothalamus was isolated for analyses. N=3-4 per group for a single RNA-seq. Heatmap represents genes upregulated by hypoxia (2h) (LFC > 0.3 and P ≤ 0.05).

(B) HOMER motif analysis of hypoxia-induced genes (start offset: −1 kb, end offset: 50 bp downstream of TSS). Enriched motifs with their name and p-value are displayed.

(C) Female C57BL/6J mice were put in normoxia or hypoxia for the indicated time points. Hypothalamus was isolated and stress GRE genes were measured via RT-qPCR. N=4-5 per group, one experiment.

(D) HIF1α and HIF2α protein levels were analysed via western blot using VINCULIN as a loading control. HIF1α and HIF2α protein levels were quantified using FIJI and normalized to VINCULIN levels. P-values were calculated using Mann-Whitney test. N=5 biological replicates per group. N = normoxia, H = hypoxia.

(E) HIF1α and HIF2α expression was detected in hypothalamus samples via IHC, scale bar 50 µm.

Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, **P<0.01, *P≤0.05.
Reprogramming of glucocorticoid receptor function by hypoxia (2021)
Figure 6

Figure 6. Hypoxia causes white adipose tissue lipolysis leading to FFA and liver ketone body production in a GR-dependent manner.

(A) Female C57BL/6J and ADX mice were put in normoxia and hypoxia for 6h and 24h. Plasma was isolated for metabolomics analysis. The heatmap represents log10 of metabolites which are significantly increased of the plasma of C57BL/6J mice (C57BL/6J P ≤ 0.05 and ADX P > 0.05, LFC > 1), significantly increased in the plasma of C57BL/6J and ADX mice (C57BL/6J P ≤ 0.05 and ADX P ≤ 0.05, LFC > 1), and significantly increased in the plasma of ADX mice only (C57BL/6J P > 0.05 and ADX P ≤ 0.05, LFC > 1).

(B) Metabolomics analysis identifying the presence of FFAs in the plasma of C57BL/6J and ADX mice 6h after hypoxia. N=6 per group, two independent experiments.

(C) PPARα gene response in the liver of C57BL/6J mice 24h after hypoxia via RT-qPCR. N=6 per group, two independent experiments.

(D-G) FFA levels and stress GRE genes were determined in the plasma and iWAT of female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (D, F), and in GRdim/dim mice and their wild-type littermates (E, G) after 6h of normoxia and hypoxia. N=4-9 per group.

(H-J) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL/6J and ADX mice (H), in female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (I), and in GRdim/dim mice and their wild-type littermates (J). N=5-9 per group.

(K) iWAT weight of C57BL/6J and ADX mice 6h after normoxia or hypoxia. N = 5-6 per group.

Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.
Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis (2019)
Figure 1

Figure 1 Decreased liver ASK1 expression in human subjects with hepatic steatosis and NASH

Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and BMI (A), plasma alanine aminotransferase (ALAT) (B) and liver fat content (C). ASK1 mRNA expression in patients with different NASH scores (D). Scatter plot and correlation coefficient (r) of ASK1 mRNA expression and ATG5 (E), ATG7 (F) and ATG12 (G) mRNA expression. Data were collected in lean subjects (n=14), obese subjects (n=23) and obese subjects with type 2 diabetes (n=19). Values are expressed as mean ± SEM (D). ***p<0.001, ****p<0.0001. Statistical tests used: Spearman correlation for A, B, C, E, F and G; ANOVA for D.
Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis (2019)
Figure 2

Figure 2 Depletion of hepatic ASK1 increases lipid storage

(A) Hepatocytes were treated with BSA, Palm or Palm+Rap for 24 h. Cells were stained for lipid droplet accumulation (Bodipy 493/503, green) and nuclei (Hoechst, blue). Lipid accumulation was quantified using automated image-based analysis (n=4 biological replicates). Scale bar represents 100 µm. (B) Shown is one representative Western blot from two independent experiments of hepatocytes transfected with siRNA targeting ASK1 (siASK1) or non-targeting siRNA control (siCtrl). (C) Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm, Palm+Rap and stained as mentioned above (n=4 biological replicates). (D) Shown is one representative Western blot from two independent experiments and quantification of LC3-II/I and p62 protein levels in siCtrl (n=4 biological replicates) or siASK1 (n=4 biological replicates) cells treated with Palm. (E) Colocalization of LC3-II punctate (red; arrow) with lipid (BODIPY 493/503, green) and nuclei (Hoechst, blue) in siCtrl or siASK1 cells treated with BSA, Palm or Palm+Rap. Scale bar represents 100 µm. (F) Colocalization of LC3-II punctate with lipid droplets (BODIPY 493/503, green) was quantified in hepatocytes transfected with siRNA targeting ASK1 (siASK1; grey bars) or non-targeting siRNA control (siCtrl; black bars) and treated with BSA or BSA+Baf for 24 hours (n=4 biological replicates). (G) Lipid accumulation was quantified using automated image-based analysis in ASK1 knockdown (siASK1; grey bars) or control (siCtrl; black bars) cells treated with BSA, Palm or Palm+Baf and stained as mentioned above (n=4 biological replicates). Values are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001. Statistical tests used: ANOVA for A, C, F and G; t-test for D.
Liver ASK1 protects from non-alcoholic fatty liver disease and fibrosis (2019)
Figure 3

Figure 3 Hepatocyte-specific ASK1-knockout mice develop NASH and fibrosis

(A) Representative images of liver sections stained with H&E from mice fed a HFD for 20 weeks. Scale bar represents 100 µm. (B) Liver triglyceride (TG) content of mice fed a chow (ASK1F/F n=6 mice; ASK1Δhep n=6 mice) or HFD (ASK1F/F n=14 mice; ASK1Δhep n=15 mice). (C) Relative mRNA expression of perilipin (Plin) and very low-density lipoprotein receptor (Vldlr) in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (Vldlr) or n=8 mice (Plin)). (D) Relative mRNA expression of genes involved in inflammation in liver of mice fed a HFD for 20 weeks (ASK1F/F n=6 mice; ASK1Δhep n=7 mice (F4/80, Mcp1) or ASK1Δhep n=8 mice (Il-6)). (E) Relative mRNA expression of collagen, type I, alpha 1 (Col1a1) (ASK1F/F n=11 mice; ASK1Δhep n=15 mice) and transforming growth factor beta 1 (Tgfβ1) (ASK1F/F n=6 mice; ASK1Δhep n=8 mice) in liver of HFD-fed mice. (F) Representative images of liver sections stained with Sirius Red (scale bar represents 100 µm) and (G) quantification of Sirius Red positive area in liver of mice fed a HFD (ASK1F/F n=8 mice; ASK1Δhep n=8 mice). (H) Representative images of liver sections stained with α-SMA from mice fed a HFD for 20 weeks. Scale bar represents 100 µm. (I) Shown is one representative Western blot from two independent experiments and quantification of hepatic COL1A1 protein levels in mice fed a HFD for 20 weeks (ASK1F/F n=8 mice; ASK1Δhep n=8 mice). (J and K) Liver triglyceride (TG) content (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) and quantification of Sirius Red positive area in livers (ASK1F/F n=7 mice; ASK1Δhep n=7 mice) of 15 months old chow-fed mice. Values are expressed as mean ± SEM. #p=0.08; *p<0.05; **p<0.01; ***p<0.001. Statistical tests used: ANOVA for B, t-test for C, D, E, G, I, J and K.