EMBO/soda-nel huggingface dataset:
Chronic cold exposure enhances glucose oxidation in brown adipose tissue (2020)
Figure 5

Fig. 5, MPC inhibition blocks oxidative metabolism in brown adipocytes and BAT.

(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats.

(B) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates.

(C) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7.

(D) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.

Data information: Data are represented as the mean ± s.d. Statistical analysis was performed using one-way ANOVA followed by Tukey multiple comparisons test (A and B), two-tailed Student's t-test (C), two-way ANOVA followed by Sidak's multiple comparisons test (D), *P < 0.05.

Expanded View figure legends
Isotope-labeled amyloid-β does not transmit to the brain in a prion-like manner after peripheral administration (2022)
Figure 3

Figure 3 - Intraperitoneally injected 13C-Lys Aβ is detectable in the periphery long after injection.

A Experimental design. i.p., intraperitoneal.

B Temporal MRM analysis of 13C-Lys Aβ17-28 and unlabeled Aβ17-28 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after 13C-Lys-APPtg brain extract i.p. injection (n = 3 mice per time point and tissue at 1-10 d, except for n = 2 mice for peritoneal cells at 3 d; n = 4 mice per time point and tissue at 25-100 d, except for n = 3 mice for spleen at 100 d). Measurements in technical triplicates, normalization to 13C15N-Lys Aβ17-28 intensity as a spiked internal standard and additionally to organ weight (mg) for lymph nodes. Error bars show median ± interquartile range.

C Temporal 4G8 immunoassay quantification of Aβ42 in the liver, spleen, mesenteric lymph nodes, and peritoneal cells after unlabeled APPtg (n = 8 mice at 5 h - 7 d, except for n = 5 mice for peritoneal lavage, n = 5 mice at 10-14 d, n = 4 mice at 21 d) or non-tg brain extract injection (n = 2 mice at 5 h and 3 d, n = 3 mice at 1 d, n = 1 mouse at 5 and 7 d, except for peritoneal cells where n = 1 mouse at 5 h and 3 d, and n = 2 mice at 1 d). Error bars show median ± interquartile range.

D Normalization of APPtg extract group data presented in (C) to organ weight (mg) and injected Aβ amount (range 1,027-1,426 ng). Error bars show median ± interquartile range.
SCFJFK Is Functionally Linked to Obesity and Metabolic Syndrome (2021)
Figure 1

Figure 1. JFK Transgenic Mice Develop Spontaneous Obesity and Are Prone to Diet-induced Metabolic Disorders.

(A) Total RNAs or proteins from tail-tip of wild-type and JFKTG mice were extracted and analyzed for Jfk expression by qPCR or western blotting with the indicated antibodies, respectively. Error bars represent mean ± SEM (n = 6, ***p < 0.001, paired two-tailed Student's t-test).

(B) Measurement of the body weight of wild-type and JFKTG mice under ND in the indicated time of age. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(C) Representative images of wild-type and JFKTG mice fed on ND. Arrows indicate abdominal SAT under MRI analysis. EWAT and PRAT were dissected, and the sections from EWAT were stained with H&E. Scale bar, 100 μm.

(D) 17-week-old wild-type or JFKTG mice fed on ND were fasted for 6-12 h prior to intraperitoneal injection of glucose or insulin. The blood was collected from the tail vein at different time points for plasma glucose measurement. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(E) The body weight of male wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(F) Representative images of wild-type or JFKTG mice fed on HFD. Arrows indicate abdominal SAT under MRI analysis. EWAT and PRAT were dissected and the sections from EWAT were stained with H&E. Scale bar, 100 μm.

(G) GTT or ITT in 17-week-old wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.
SCFJFK Is Functionally Linked to Obesity and Metabolic Syndrome (2021)
Figure 2

Figure 2. JfkKO Mice Are Resistant to Diet-induced Obesity and Hepatic Steatosis.

(A) Total RNAs or proteins from tail-tip of wild-type or JfkKO mice were extracted and analyzed for Jfk expression by qPCR or western blotting with the indicated antibodies, respectively. Error bars represent mean ± SEM (n = 6, ***p < 0.001, paired two-tailed Student's t-test).

(B) The body weight of wild-type or JfkKO mice under ND were measured at the indicated time of age. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(C) Representative images of wild-type or JfkKO mice fed on ND. Arrows indicate abdominal SAT under MRI. EWAT and PRAT were dissected, and the sections from EWAT were stained with H&E. Scale bar, 100 μm.

(D) The body weight of male wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(E) Representative images of wild-type or JfkKO mice fed on HFD. Arrows indicate abdominal SAT under MRI. EWAT and PRAT were dissected, and the sections from EWAT were stained with H&E. Scale bar, 100 μm.

(F) GTT or ITT in 17-week-old wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm.

(H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm.

(I) Wild-type, JfkKO, or JfkKO mice with liver-specific reconstitution of JFK were housed with free access to water but no food during indirect calorimetry. The white or gray horizontal bars depict the light or dark period in a 12: 12 h light-dark cycle, respectively. EE or RER was calculated and plotted at 1 h intervals and averaged. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test).
SCFJFK Is Functionally Linked to Obesity and Metabolic Syndrome (2021)
Figure 3

Figure 3. JFK Is Physically Associated with ING5 in Hepatocytes.

(A) HepG2 cells were stably transfected with vector or FLAG-JFK. Cellular extracts were immunopurified with anti-FLAG immunoafﬁnity resin and eluted with FLAG peptides. The eluates were resolved on SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry.

(B) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins.

(C) Extracts from liver tissues of wild-type mice were immunoprecipitated with antibodies against Ing5 followed by immunoblotting with the antibodies against the indicated proteins.

(D) GST pull-down assays with bacterially expressed GST-ING5 and in vitro transcribed/translated JFK, CUL1, SKP1, or RBX1.

(E) Schematic diagrams of wild-type JFK and JFK mutants. Cellular extracts from HepG2 cells co-transfected with Myc-ING5 and FLAG-JFK or JFK mutants were immunoprecipitated with anti-FLAG followed by immunoblotting with anti-Myc.

(F) GST pull-down assays with bacterially expressed GST-JFK or JFK mutants and in vitro transcribed/translated ING5.
SCFJFK Is Functionally Linked to Obesity and Metabolic Syndrome (2021)
Figure 4

Figure 4. SCFJFK Targets ING5 for Degradation.

(A) Western blotting analysis of cellular extracts from HepG2 cells transfected with FLAG-JFK or JFK mutants.

(B) Western blotting analysis of cellular extracts from HepG2 cells transfected with the indicated plasmids.

(C) HepG2 cells treated with control siRNAs or siRNAs against CDC20, CUL1, or SKP1 were transfected with vector or FLAG-JFK. Cellular extracts were prepared for western blotting analysis. Total RNAs were extracted and analyzed for ING5 expression by qPCR. Error bars represent mean ± SD for triplicate experiments.

(D) HepG2 cells were transfected with FLAG-JFK and treated with MG132 prior to protein extraction for western blotting analysis.

(E) HepG2 cells were transfected with FLAG-JFK and treated with 50 μg/ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING5/β-actin.

(F) Cellular extracts from HCT116 p53+/+ and HCT116 p53-/- cells transfected with empty vector or FLAG-JFK were prepared for western blotting analysis.

(G) HepG2 cells were co-transfected with FLAG-tagged JFK, JFK∆F-box, or JFK∆Kelch, together with HA-Ub or UbK48R, and treated with 5 μM MG132 for 12 h prior to in vivo ubiquitination assays with the indicated antibodies.

(H) Bacterially-expressed GST-JFK or JFK mutants was incubated with in vitro transcribed/translated ING5 for in vitro ubiquitination assays.
SCFJFK Is Functionally Linked to Obesity and Metabolic Syndrome (2021)
Figure 5

Figure 5. Genome-wide Identification of Transcriptional Targets for SCFJFK-ING5 Axis.

(A) Genomic distribution of ING5 determined by ChIP-seq analysis. ChIP was performed in HepG2 cells with antibodies against ING5 followed by deep sequencing. The raw data were analyzed by MACS with the cutoff of p value < 1e-5 and FDR < 0.001.

(B) Binding density of ING5 across its specific binding sites visualized by deepTools. The heatmap shows the normalized ChIP-seq tag counts ordered by signal strength.

(C) Venn diagram from cross analysis of down-regulated genes obtained from RNA-seq analysis in JFK-overexpressing and ING5-depleted HepG2 cells with genes targeted by ING5 from ChIP-seq. GO enrichment analysis of 140 potential target genes using DAVID is shown.

(D) ChIP-seq track at the representative promoter of target genes. The red rectangles indicate the peak regions/binding sites of ING5 on the target genes promoter.
SCFJFK Is Functionally Linked to Obesity and Metabolic Syndrome (2021)
Figure 6

Figure 6. The SCFJFK-ING5 Axis Intersects with AMPK Activity and Fatty-acid β-oxidation to Regulate Hepatic Lipid Metabolism.

(A) HepG2 cells were transfected with control or ING5, or treated with siRNAs against JFK. Soluble chromatin was prepared and qChIP analysis was performed on the selected promoters using the indicated antibodies. Histone H3 was detected as an internal control. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).

(B) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector or JFK or/and ING5. Total RNAs were extracted and qPCR analysis was performed. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).

(C) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector, JFK, or/and ING5. Cellular extracts were prepared for western blotting analysis with the indicated antibodies.

(D) HepG2 cells were transfected with vector or JFK, AMPKα1, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5, Cellular extracts were prepared for AMPK activity assays by ELISA. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).

(E) HepG2 cells were transfected with vector, JFK, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5. Mitochondria were isolated and the rate of fatty acid β-oxidation was measured by spectrophotometry of ferricyanide-trapped reduced products. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).

(F) HepG2 cells were transfected with vector, JFK, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5. Culture media was collected and the concentration of 3-hydroxybutyric acid and acetoacetic acid was measured by ketone body assay kit. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).
SCFJFK Is Functionally Linked to Obesity and Metabolic Syndrome (2021)
Figure 7

Figure 7. Pathophysiological Significance of the SCFJFK-ING5 Axis.

(A) Western blotting analysis of mouse tissues for the expression of Jfk and Ing5.

(B and C) Total proteins or RNAs were prepared in MEFs or liver tissues from wild-type or JfkKO mice for western blotting analysis (B) or qPCR measurement (C), respectively. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).

(E and F) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown (F). Scale bar, 100 µm.

(G) Analysis of public dataset (GSE135251) for JFK and AMPKα1 mRNA expression in liver tissues from healthy controls (n = 10) and NAFLD patients (n = 206). Whiskers indicate upper or lower quartile, and lines inside the box represent median (*p < 0.05, welch two sample t-test).

(H) The proposed model of the SCFJFK-ING5 axis in the development of obesity and metabolic syndrome.

EXPANDED VIEW (EV) FIGURE LEGENDS
C9ORF72 loss synergizes DPR proteins toxicity, a mechanism prevented by drugs activating autophagy (2019)
Figure 1

Figure 1. Expanded G4C2 repeats are translated into polyGA and polyGR.

(A) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 and fused in all three possible frames with the GFP deleted of its ATG.

(B) LC-MS/MS spectra of the N-terminal part of the GFP-immunoprecipitated and LysC digested polyGA protein expressed as in (A).

(C, D) Immunoblotting against the GFP (C) or the HA tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3, 20 or 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame.

(E) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected with either a wild type or mutant (AGG into CGG) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GR frame.

(F) Scheme of human C9ORF72 sense transcript with intron 1 retained. Expanded G4C2 repeats, CUG and AGG near-cognate initiation codons, polyGA and polyGR ORFs are indicated in red. C9ORF72 ORF is indicated in blue.
C9ORF72 loss synergizes DPR proteins toxicity, a mechanism prevented by drugs activating autophagy (2019)
Figure 2

Figure 2. Expanded C4G2 repeats are translated into polyPG.

(A) Immunoblotting against the HA tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 and fused in all three possible frames with a HA tag.

(B) LC-MS/MS spectra of the N-terminal part of the HA-immunoprecipitated and LysC digested polyPG protein expressed as in (A).

(C, D) Immunoblotting against the GFP (C) or the HA tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3, 10 or 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to the GFP in the PG frame.

(E) Scheme of human antisense C9ORF72 transcript. Expanded C4G2 repeats, AUG initiation codon and polyPG ORF are indicated in red.
C9ORF72 loss synergizes DPR proteins toxicity, a mechanism prevented by drugs activating autophagy (2019)
Figure 3

Figure 3. Decreased expression of C9ORF72 synergizes DPR proteins toxicity.

(A) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.

(B) Immunoblotting against the GFP, endogenous C9ORF72 or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA.

(C) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.

(D) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.

(E) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild-type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.

Data information: Error bars indicate s.e.m. Student's T-test, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001. n=5 independent transfection.
C9ORF72 loss synergizes DPR proteins toxicity, a mechanism prevented by drugs activating autophagy (2019)
Figure 4

Figure 4. Promethazine reduces DPR proteins accumulation and toxicity.

(A) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyGA expression relative to the GAPDH.

(B) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyPG expression relative to the GAPDH.

(C) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells treated with 1, 3 or 10 µM of promethazine and co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.

Data information: Error bars indicate s.e.m. Student's T-test, ** indicates p<0.01, *** indicates p<0.001. n=5 independent transfection.
Oligonucleotide-induced alternative splicing of serotonin 2C receptor reduces food intake (2016)
Figure 1

Figure 1: Identification of oligonucleotides that change alternative splicing of the serotonin 2C receptor

A. Pre-mRNA processing of the serotonin 2C receptor. Exons are shown as boxes, introns are lines. Due to the use of two alternative splice sites (PS, DS: proximal and distal splice site) exon Vb is alternatively used, resulting in two RNAs: RNA1 lacking and RNA2 including exon Vb. Alternative splicing patterns are indicated by dashed and dotted lines; constitutive splicing patterns are indicated by solid lines. The start codon is indicated using a line with circle. Alternative usage of exon Vb introduces a frameshift, leading to different stop codons being used (lines with rectangles in exon VI, red box in exon VI indicates a different reading frame). The proteins formed by RNA1 (truncated 5HT2C) and RNA2 (full-length 5HT2C) are indicated. Circles in the second intracellular loop of RNA2-protein depict three amino acids changed by RNA editing. The location of the epitope against the truncated 5HT2C is indicated with a Y.

B. The truncated receptor encoded by RNA1 is endogenously expressed. HEK293 cells were transfected with a cDNA encoding the human truncated 5HT2C (RNA1). SDS-lysates from these transfected cells and from mouse hypothalamus were analyzed by Western blot using an antiserum made against RNA1 (Figure EV1). Mouse hypothalamus was exposed 10 times longer. In brain, a band around 75kDa of unknown origin is detected by the antiserum (indicated by a star).

C. The minigene used for the oligo-walk is schematically shown; the arrow indicates the CMV promoter. The boxed area corresponds to the sequence shown below. The secondary structure of the serotonin 2C receptor pre-mRNA determined by SHAPE (Shen et al, 2013) is shown and the oligonucleotides used are indicated as lines. The binding site for SNORD115 (Kishore & Stamm, 2006) is boxed and five adenosines undergoing RNA editing are highlighted. The exon/alternative exon border in the proximal splice site (GG) and the alternative exon/intron border (Gg, distal splice site) are underlined and marked in red. Dots indicate base pairing, including non-canonical G-U base pairs.

D. RT-PCR analysis of the serotonin 2C receptor minigene after oligonucleotide addition. Below is a quantification of three independent experiments. A statistical evaluation showed significant differences between oligo#5 (p=0.00008) and oligo#5-3 (p= 0.0019) and control.